22012, a novel human carboxypeptidase

ABSTRACT

The present invention relates to a newly identified human carboxypeptidase. The invention also relates to polynucleotides encoding the carboxypeptidase. The invention further relates to methods using the carboxypeptidase polypeptides and polynucleotides as a target for diagnosis and treatment in carboxypeptidase-related disorders. The invention further relates to drug-screening methods using the carboxypeptidase polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and treatment. The invention further encompasses agonists and antagonists based on the carboxypeptidase polypeptides and polynucleotides. The invention further relates to procedures for producing the carboxypeptidase polypeptides and polynucleotides.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.09/345,469, filed Jun. 30, 1999, which is hereby incorporated in itsentirety by reference herein.

FIELD OF THE INVENTION

[0002] The present invention relates to a newly identified humancarboxypeptidase. The invention also relates to polynucleotides encodingthe carboxypeptidase. The invention further relates to methods using thecarboxypeptidase polypeptides and polynucleotides as a target fordiagnosis and treatment in carboxypeptidase-related disorders. Theinvention further relates to drug-screening methods using thecarboxypeptidase polypeptides and polynucleotides to identify agonistsand antagonists for diagnosis and treatment. The invention furtherencompasses agonists and antagonists based on the carboxypeptidasepolypeptides and polynucleotides. The invention further relates toprocedures for producing the carboxypeptidase polypeptides andpolynucleotides.

BACKGROUND OF THE INVENTION

[0003] Proteolytic enzymes are involved in many cellular processes. Thecarboxypeptidase family of enzymes catalyzes the cleavage of C-terminalamino acids of peptides and proteins, altering their biologicalactivity. Lysosomal carboxypeptidase enzymes are highly concentrated inlysosomes, but may also be active extracellularly after their releasefrom lysosomes in soluble form or bound to transmembrane or othermembrane-associated proteins. Carboxypeptidases may cleave peptides in asequence-specific manner. For example, prolylcarboxypeptidases cleaveonly peptides linked to proline residues (for example,des-Arg9-bradykinin, angiotensin II). There is also evidence that theseenzymes are involved in terminating signal transduction by inactivatingpeptide ligands after receptor endocytosis.

[0004] In contrast to endoproteases which cleave internal peptide bondsof proteins and polypeptides, carboxypeptidases (CPs) catalyze thecleavage of only the C-terminal peptide bond, releasing one amino acidat a time. The two main groups of CPs include serine CPs andmetallo-CPs, the serine CPs containing a signature trio of Ser, Asp, Hisin the active site. This trio is also contained in prolylendopeptidaseserine proteases. Serine CPs include polycarboxypeptidase (PRCP) alsoreferred to as angiotensinase C; and deamidase, also referred to ascathepsin A and lysosomal protective protein. See Skidgel et al. (1998)Immunological Reviews 161:129-141.

[0005] Metallo-CPs contain a signature glutamic acid as the primarycatalytic residue and require zinc-binding for activity. Metallo-CPs canbe grouped by substrate specificity into CPA and CPB types; the CPA typepreferentially cleaving C-terminal hydrophobic residues, and the CPBtype cleaving only peptides with C-terminal basic Arg or Lys residues.See R. A. Skidgel (1993) In: Hooper NM, ed. Zinc Metalloproteases inHealth and Disease, London: Taylor & Francis, Ltd., p. 241-283.

[0006] CPM is a B type carboxypeptidase which is anchored on cellmembranes via gylcosylphosphatidylinositol (GPI) association with itsmildly hydrophobic stretch of 15 C-terminal amino acids. As in manyother proteins sharing this anchoring mechanism, CPM is released fromthe membrane by bacterial phosphatidylinositol-specific phospholipase C.Human CPM is a glycoprotein of 426 amino acid residues with 43% identityto human intracellular secretory granular CP (CPE), 41% with the active50 kDa subunit of human plasma CPN, and 15% with bovine pancreatic CPAor CPB. The active sites of these CPs contain conserved amino acidresidues corresponding to the zinc binding residues His⁶⁶Glu⁶⁹ andHis¹⁷³, substrate binding residues Arg¹³⁷ and Tyr²⁴², and the catalyticGlu²⁶⁴, as designated for CPM. Sequence homologies around theseconserved residues is high, with an identity between CPs M, E and N ofapproximately 70-90%. See Tan et al. (1989) J. Biol. Chem.264:13165-13170; Deddish et al. (1990) J. Biol. Chem. 265:15083-15089;R. A. Skidgel (1993) In: Hooper NM, ed. Zinc Metalloproteases in Healthand Disease, London: Taylor & Francis, Ltd., p. 241-283. CPM has beenmapped to the chromosomal location of chromosome 12q13-q15 which isassociated with a variety of solid tumors.

[0007] The optimal pH range of CPM is in the neutral range of 6.5-7.5.As no endogenous inhibitors are known for CPM, the enzyme is consideredto be constitutively active. Synthetic inhibitors including Arg analogsDL-2 mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA) andguanidinoethylmercaptosuccinic acid (GEMSA) inhibit CPM. See R. A.Skidgel (1991) In: Conn PM, ed. Methods in Neurosciences: PeptideTechnology Vol. 6, Orlando: Academic Press, p. 373-385; Plummer et al.(1981) Biochem. Biophys. Res. Comm. 98: 448-254.

[0008] As with other B type regulatory CPs, CPM cleaves only C-terminalArg or Lys residues; however, CPM has a preference for the C-terminalArg. The penultimate amino acid also affects the rate of hydrolysis.Naturally occurring peptide substrates of CPM include bradykinin, Arg⁶-and Lys⁶ enkephalins, dynorphin A¹⁻¹³ and epidermal growth factor (EGF).See Sidgel et al. (1989) J. Biol. Chem. 264:2236-2241; McGwire et al.(1995) J. Biol. Chem. 270:17154-17158.

[0009] CPM is primarily found on the plasma membrane, with highestlevels found in lung and placenta. It is also present in kidney, bloodvessels, intestine, brain and peripheral nerves. See R. A. Skidgel(1988) Trends Pharm. Sci. 9:299-304; Skidgel et al. (1984) Biochem.Pharmacol. 33: 3471-3478; Skidgel et al. (1991) FASEB J. 5: 1578; Nagaeet al. (1992) J. Neurochem. 59:2201-2212; Nagae et al. (1993) Am. J.Respir. Cell Mol. Biol. 9:221-229. Expression of CPM is responsive todifferentiation of monocytes and lymphocytes. See de Saint-Vis et al.(1995) Blood 86:1098-1105; Rehli et al. (1995) J. Biol. Chem.270:15644-15649.

[0010] CPM participates in the control of peptide hormone activity atthe cell surface and degradation of extracellular proteins and peptides.It catalyzes the second step in prohormone processing and removesC-terminal Arg or Lys residues from peptides released from prohormones.CPM functions as a soluble enzyme after its release from the plasmamembrane and may function in the plasma membrane form to control peptidereceptor activities. CPM can regulate receptor specificity of kinins bycleaving the C-terminal ARG⁹, for example, from bradykinin. The intactbradykinin binds the B2 receptor. The cleaved bradykinin(des-ARG⁹-bradykinin). Des-ARG⁹-bradykinin also binds the B1 receptors:stimulates IL-1 and tumor necrosis factor release from macrophages.Regulation of the B1 receptor is associated with injury or inflammation.CPM may also be involved with other inflammatory mediators, such asanaphylatoxin C5a which mediates histamine release. In addition, CPM maymetabolize growth factors containing terminal Arg or Lys, such as EGF,EGF-like peptides, nerve growth factor (NGF) amphiregulin, hepatocytegrowth factor, erythropoietin, and macrophage-stimulating protein. Inthe lung, varying levels of CPM are associated with pneumocystic orbacterial pneumonia or lung cancer, and in the placenta, CPM may protectthe fetus from maternally derived peptides. See R. A. Skidgel (1992) J.Cardiovasc. Pharmacol. 20(Suppl. 9):S4-S9; Bhoola et al. (1992)Pharmacol. Rev. 44:1-80; R. A. Skidgel (1993) In: Hooper NM, ed. ZincMetalloproteases in Health and Disease, London: Taylor & Francis, Ltd.,p. 241-283; Dragovic et al. (1995) Am. J. Respir. Crit. Care Med.152:760-764; Nagae et al. (1992) J. Neurochem. 59:2201-2212; MacFaddenet al. (1988) FASEB J. 2:1179 (Abstract).

[0011] Another B-type regulatory CP metalloprotein is CPD, amembrane-bound glycoprotein. Human CPD is a protein of 1,377 amino acidswith 75% identity with duck GP180 and 90% identity with rat CPD. HumanCPD contains two hydrophobic regions located at the C- and N-termini. A55-60 residue cytoplasmic domain is highly conserved among duck, humanand rat sequences and may be significant in intracellular sorting,protein-protein interactions or endocytosis. CPD contains three tandemCP homology domains numbered sequentially from the N- to the C-terminus,and thereby may contain more than one active site. See Tan et al. (1997)Biochem. J. 327:81-87; Skidgel et al. (1993) In: Robertson JLS NichollsMG, eds. The Renin Angiotensin System, Vol 1, London: Gower MedicalPublishing, p. 10.1-10.10. CPD is located on human chromosome 17, 17P,11.1-17q, 11.2.

[0012] CPD is primarily found on intracellular membranes, mainly in theGolgi, with some CPD found on the plasma membrane. The tissuedistribution of CPD is wide and includes most duck tissues and mammaliantissues as well, including brain, pituitary, placenta, pancreas,adrenal, kidney, lung, heart, spleen, intestine, ovary, and testes. SeeMcGwire et al. (1997) Life Sci. 60:715-724; Song et al. (1995) J. Biol.Chem. 270:25007-25013; Xin et al. (1997) DNA Cell Biol. 16:897-909; Tanet al. (1997) Biochem. J. 327:81-87; Song et al. (1996) J. Biol. Chem.271:28884-28889.

[0013] The function of CPD is speculated to include peptide and proteinprocessing in the constitutive secretory pathway after endoproteasecleavage of precursor proteins. The enzyme has an acidic pH optimum.Mammalian CPD may act as a hepatitis B virus binding protein, similar tothe duck CPD. See R. A. Skidgel (1998) Immunological Reviews161:129-141.

[0014] Serine CPs include PRCP and deamidase. PRCP cloned from a humankidney library indicates a glycoprotein of 51 kDa³; and containing 496amino acids, including a 30 residue signal peptide and a 15 residuepropeptide. See Tan et al. (1993) J. Biol. Chem. 268:16631-16638. Aserine repeat is found in the C-terminal half, similar to the serinerepeat of a yeast CP encoded by the KEX1 gene.

[0015] PRCP has an acidic pH optimum for synthetic peptide substrates,but retains activity at neutral ranges with longer naturally occurringpeptides. PRCP cleaves peptides only if the penultimate residue isproline. The enzyme does not cleave Pro-Pro-COOH or (OH)-Pro-Pro-COOHbond. See Odya et al. (1978) J. Biol. Chem. 253:5927-5931. Substrates ofPRCP include des-Arg⁹-bradykinin and angiotensin II.

[0016] PRCP may be involved in terminating signal transduction byinactivating peptide ligands after receptor endocytosis. PRCP iscontained in lysosomes and released in response to stimulation. Theenzyme is widely distributed and found in human placenta, lung, liver,and kidney.

[0017] Another serine CP, deamidase, is likely a 94 kDa homodimer of 52kDa subunits. Human platelet deamidase is activated by cleavage of a 14amino acid fragment from the C-terminus. The enzyme binds and maintainsactivity and stability of β-galactocidase and neuraminidase inlysosomes, a defect of which is associated with severegalactosialidosis. See Bonten et al. (1995) J. Biol Chem.270:26441-26445; Galjart et al. (1988) Cell 54:755-764; D'Azzo et al.(1982) Proc. Natl. Acad. Sci. 79:4535-4539. The gene for the humandeamidase is mapped to chromosome 20 at q13.1.

[0018] Deamidase cleaves various peptides containing C-terminal orpenultimate hydrophobic residues including substance P, angiotensin I,bradykinin, endothelin, and fMet-Leu-Phe. Like PRCP, deamidase is alsofound in lysosomes, and distributed in human placenta, lung, liver, andkidney. Like PRCP, deamidase is implicated in blocking part of thesignal transduction pathway stimulated by peptides. Bradykinin,containing a C-terminal Arg⁹ and a penultimate hydrophobic amino acidPhe⁸, is cleaved by deamidase. Similarly, angiotensin, containing aC-terminal His and a penultimate Phe, is cleaved by deamidase.Accordingly, deamidase is implicated in termination of bradykininactivity on the B2 receptor to generate a B1 receptor agonist. Deamidasemay also have a role in chemotaxis and in metabolism of the anti-cancergrowth factor antagonist. See Skidgel et al. (1998) ImmunologicalReviews 161:129-141; Jackman et al (1990) J. Biol. Chem.265:11265-11272; Jackman et al. (1995) Am. J. Respir. Cell Mol. Biol.13:196-204; Hinek et al. (1996) Biol. Chem. 377:471-480; Jones et al.(1995) Peptides 16:777-783; Cummings et al. (1995) Biochem Pharmacol.49:1709-1712.

[0019] Given the wide distribution and various physiological andpathological roles of carboxypeptidases, methods and compositionsdirected at regulating levels of these enzymes are useful for regulatingpeptide hormone activity, modulating metabolism of substance P,angiotensin I, angiotensin II, bradykinin, and endothelin, andregulation of signal transduction by inactivation of peptide ligandssubsequent to receptor endocytosis.

[0020] Accordingly, carboxypeptidases are a major target for drug actionand development. Therefore, it is valuable to the field ofpharmaceutical development to identify and characterize previouslyunknown carboxypeptidases. The present invention advances the state ofthe art by providing a previously unidentified human carboxypeptidase.

SUMMARY OF THE INVENTION

[0021] It is an object of the invention to identify novelcarboxypeptidases.

[0022] It is a further object of the invention to provide novelcarboxypeptidase polypeptides that are useful as reagents or targets incarboxypeptidase assays applicable to treatment and diagnosis ofcarboxypeptidase-related disorders.

[0023] It is a further object of the invention to providepolynucleotides corresponding to the novel carboxypeptidase polypeptidesthat are useful as targets and reagents in carboxypeptidase assaysapplicable to treatment and diagnosis of carboxypeptidase-relateddisorders and useful for producing novel carboxypeptidase polypeptidesby recombinant methods.

[0024] A specific object of the invention is to identify compounds thatact as agonists and antagonists and modulate the expression of the novelcarboxypeptidase.

[0025] A further specific object of the invention is to providecompounds that modulate expression of the carboxypeptidase for treatmentand diagnosis of carboxypeptidase-related disorders.

[0026] The invention is thus based on the identification of a novelhuman carboxypeptidase. The amino acid sequence is shown in SEQ IDNO: 1. The nucleotide sequence is shown as SEQ ID NO:2.

[0027] The invention provides isolated carboxypeptidase polypeptides,including a polypeptide having the amino acid sequence shown in SEQ IDNO:1 or the amino acid sequence encoded by the cDNA deposited as ATCCNo. PTA-1643 on Apr. 5, 2000 (“the deposited cDNA”).

[0028] The invention also provides isolated carboxypeptidase nucleicacid molecules having the sequence shown in SEQ ID NO:2 or in thedeposited cDNA.

[0029] The invention also provides variant polypeptides having an aminoacid sequence that is substantially homologous to the amino acidsequence shown in SEQ ID NO:1 or encoded by the deposited cDNA.

[0030] The invention also provides variant nucleic acid sequences thatare substantially homologous to the nucleotide sequence shown in SEQ IDNO:2 or in the deposited cDNA.

[0031] The invention also provides fragments of the polypeptide shown inSEQ ID NO:1 and nucleotide sequence shown in SEQ ID NO:2, as well assubstantially homologous fragments of the polypeptide or nucleic acid.

[0032] The invention further provides nucleic acid constructs comprisingthe nucleic acid molecules described herein. In a preferred embodiment,the nucleic acid molecules of the invention are operatively linked to aregulatory sequence.

[0033] The invention also provides vectors and host cells for expressingthe carboxypeptidase nucleic acid molecules and polypeptides, andparticularly recombinant vectors and host cells.

[0034] The invention also provides methods of making the vectors andhost cells and methods for using them to produce the carboxypeptidasenucleic acid molecules and polypeptides.

[0035] The invention also provides antibodies or antigen-bindingfragments thereof that selectively bind the carboxypeptidasepolypeptides and fragments.

[0036] The invention also provides methods of screening for compoundsthat modulate expression or activity of the carboxypeptidasepolypeptides or nucleic acid (RNA or DNA).

[0037] The invention also provides a process for modulatingcarboxypeptidase polypeptide or nucleic acid expression or activity,especially using the screened compounds. Modulation may be used to treatconditions related to aberrant activity or expression of thecarboxypeptidase polypeptides or nucleic acids.

[0038] The invention also provides assays for determining the activityof or the presence or absence of the carboxypeptidase polypeptides ornucleic acid molecules in a biological sample, including for diseasediagnosis.

[0039] The invention also provides assays for determining the presenceof a mutation in the polypeptides or nucleic acid molecules, includingfor disease diagnosis.

[0040] In still a further embodiment, the invention provides a computerreadable means containing the nucleotide and/or amino acid sequences ofthe nucleic acids and polypeptides of the invention, respectively.

DESCRIPTION OF THE DRAWINGS

[0041]FIG. 1 shows the carboxypeptidase nucleotide sequence (SEQ IDNO:2) and the deduced amino acid sequence (SEQ ID NO:1).

[0042]FIG. 2 shows a comparison of the carboxypeptidase against theProsite database of protein patterns, specifically showing a high scoreagainst the prolyloligopeptidase family (SEQ ID NO:3) and an alpha/betahydrolase (SEQ ID NO:4).

[0043]FIG. 3 shows an analysis of the carboxypeptidase amino acidsequence: αβturn and coil regions; hydrophilicity; amphipathic regions;flexible regions; antigenic index; and surface probability plot.

[0044]FIG. 4 shows a hydrophobicity plot of the carboxypeptidase (SEQ IDNO:1).

[0045]FIG. 5 shows an analysis of the carboxypeptidase open readingframe for amino acids corresponding to specific functional sites.Glycosylation sites, protein kinase C phosphorylation sites, caseinkinase II phosphorylation sites, N-myristoylation sites, and leucinezipper pattern of SEQ ID NO:1 are shown.

[0046]FIG. 6 shows RNA expression of the carboxypeptidase in normal andtumor tissues.

[0047]FIG. 7 shows RNA expression of the carboxypeptidase in normalhuman tissues.

DETAILED DESCRIPTION OF THE INVENTION

[0048] Polypeptides

[0049] The invention is based on the discovery of a novel humancarboxypeptidase. Specifically, an expressed sequence tag (EST) wasselected based on homology to carboxypeptidase sequences. This EST wasused to design primers based on sequences that it contains and used toidentify a cDNA found in osteoblast, brain, small intestine, heart, andprostate cDNA libraries. Positive clones were sequenced and theoverlapping fragments were assembled. Analysis of the assembled sequencerevealed that the cloned cDNA molecule encodes a carboxypeptidase.

[0050] The invention thus relates to a novel carboxypeptidase having thededuced amino acid sequence shown in FIG. 1 (SEQ ID NO: 1) or having theamino acid sequence encoded by the deposited cDNA, ATCC No. PTA-1643.

[0051] The deposit will be maintained under the terms of the BudapestTreaty on the International Recognition of the Deposit ofMicroorganisms. The deposit is provided as a convenience to those ofskill in the art and is not an admission that a deposit is requiredunder 35 U.S.C. § 112. The deposited sequence, as well as thepolypeptide encoded by the sequence, is incorporated herein by referenceand controls in the event of any conflict, such as a sequencing error,with description in this application.

[0052] The carboxypeptidase of the invention has homology to the familyof prolylendopeptidases. The catalytic triad signature, SER ASP HIS, isfound in the protein of the invention as well as inprolylcarboxypeptidase (angiotensinase C NOBF [P42785], PCP). PCP isclassified as belonging to the prolylendopeptidase and serinecarboxypeptidase family. The amino acids around the SER in PCP isconsistent with a prolylendopeptidase. The ones found in the polypeptideof the invention are more similar to the ones found in the serinecarboxypeptidase family.

[0053] “Carboxypeptidase polypeptide” or “carboxypeptidase protein”refers to the polypeptides in SEQ ID NO:1 or encoded by the depositedcDNA. The term “carboxypeptidase protein” or “carboxypeptidasepolypeptide”, however, further includes the numerous variants describedherein, as well as fragments derived from the full-lengthcarboxypeptidase and variants.

[0054] Tissues and/or cells in which the carboxypeptidase is foundinclude, but are not limited to, those found in the figures herein. Inparticular, the carboxypeptidase is expressed in prostate, breast,skeletal muscle, brain, testis, thyroid, fetal kidney, fetal liver, andfetal heart tissues. Disease expression is associated with coloncarcinoma, breast carcinoma, and lung squamous cell carcinoma.Expression has been observed in HCT116, a variant cell line isolatedfrom a colon carcinoma, HT29, an adenocarcinoma line, KM12, a weaklymetastatic colorectal carcinoma cell line, and HTC8, a colon carcinomacell line. Expression has also been observed in the breast carcinomacell line MDA-231, MCF-7, HMEC, ZR-75, and MDA-435. Up-regulation hasbeen observed in HCT116, DLD-1 (adenocarcinoma of the sigmoid colon),HT29, SW480 (adenocarcinoma from metastatic lymph node), SW620(adenocarcinoma from metastatic lymph node), and KMI2. Thus, thiscarboxypeptidase is overexpressed in a number of breast, lung and colontumors, including colon carcinoma, lung adenocarcinoma, small cell lungcarcinoma, and colon metastatic tissue. Elevated levels in clinicalcancerous tumors and not in noncancerous normal tissues indicate acellular proteolytic imbalance in these tissues. Accordingly, expressionof the carboxypeptidase is relevant to carcinogenesis, includinginvasion and metastasis.

[0055] The present invention thus provides an isolated or purifiedcarboxypeptidase polypeptide and variants and fragments thereof.

[0056] Based on a BLAST search, highest homology was shown to a humanprolylcarboxypeptidase.

[0057] As used herein, a polypeptide is said to be “isolated” or“purified” when it is substantially free of cellular material when it isisolated from recombinant and non-recombinant cells, or free of chemicalprecursors or other chemicals when it is chemically synthesized. Apolypeptide, however, can be joined to another polypeptide with which itis not normally associated in a cell and still be considered “isolated”or “purified.”

[0058] The carboxypeptidase polypeptides can be purified to homogeneity.It is understood, however, that preparations in which the polypeptide isnot purified to homogeneity are useful and considered to contain anisolated form of the polypeptide. The critical feature is that thepreparation allows for the desired function of the polypeptide, even inthe presence of considerable amounts of other components. Thus, theinvention encompasses various degrees of purity.

[0059] In one embodiment, the language “substantially free of cellularmaterial” includes preparations of the carboxypeptidase having less thanabout 30% (by dry weight) other proteins (i.e., contaminating protein),less than about 20% other proteins, less than about 10% other proteins,or less than about 5% other proteins. When the polypeptide isrecombinantly produced, it can also be substantially free of culturemedium, i.e., culture medium represents less than about 20%, less thanabout 10%, or less than about 5% of the volume of the proteinpreparation.

[0060] A carboxypeptidase polypeptide is also considered to be isolatedwhen it is part of a membrane preparation or is purified and thenreconstituted with membrane vesicles or liposomes.

[0061] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the carboxypeptidase polypeptide inwhich it is separated from chemical precursors or other chemicals thatare involved in its synthesis. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of the polypeptide having less than about 30% (by dryweight) chemical precursors or other chemicals, less than about 20%chemical precursors or other chemicals, less than about 10% chemicalprecursors or other chemicals, or less than about 5% chemical precursorsor other chemicals.

[0062] In one embodiment, the carboxypeptidase polypeptide comprises theamino acid sequence shown in SEQ ID NO:1. However, the invention alsoencompasses sequence variants. Variants include a substantiallyhomologous protein encoded by the same genetic locus in an organism,i.e., an allelic variant. The carboxypeptidase has been mapped to humanchromosome 9 at 9q33-34. Nearby known genes include DYT1, CAIN, VAV2,NOTCH1, ABL1, SDHD, TSC1, SURF1, RPL7A, RXRA, COL5A1, PAEP, ABC2, N14,LCN1, CACNA1B, FCN1. Nearby mutations/loci include Human-DYS,DYSAUTONOMIA, FAMILIAL; MUSCULAR DYSTROPHY, LIMB-GIRDLE, TYPE 2H; AFD1,ACROFACIAL DYSOSTOSIS 1, BDB1, NAGER TYPE; BRACHYDACTYLY, TYPE B1; ALS4,AMYOTROPHIC LATERAL SCLEROSIS 4, JUVENILE.

[0063] A possible locus in the mouse is on chromosome 2 and possiblychromosome 4. In the mouse, the Scc2 locus controls susceptibility to1,2 dimethylhydrazine-induced colon tumors. The following loci in themouse are relevant: Mouse Chr2- Scc2, colon tumor susceptibility 2; stu,stumbler; Sd, Danforth's short tail; stb, stubby; us, urogenitalsyndrome; ebo, ebouriffe; sar, sarcosinemia autosomal recessive; Lsr1,listeria resistance; Anth2, resistance to Bacillus anthracis 2; mdm,muscular dystrophy with myositis. See Moen, C. J. et al. Proc. Natl.Acad. Sci. U.S.A. 93 (1996).

[0064] Variants also encompass proteins derived from other genetic lociin an organism, but having substantial homology to the carboxypeptidaseof SEQ ID NO:1. Variants also include proteins substantially homologousto the carboxypeptidase but derived from another organism, i.e., anortholog. Variants also include proteins that are substantiallyhomologous to the carboxypeptidase that are produced by chemicalsynthesis. Variants also include proteins that are substantiallyhomologous to the carboxypeptidase that are produced by recombinantmethods. It is understood, however, that variants exclude any amino acidsequences disclosed prior to the invention.

[0065] As used herein, two proteins (or a region of the proteins) aresubstantially homologous when the amino acid sequences are at leastabout 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, typically at least about80-85%, and most typically at least about 90-95% or more homologous. Asubstantially homologous amino acid sequence, according to the presentinvention, will be encoded by a nucleic acid sequence hybridizing to thenucleic acid sequence, or portion thereof, of the sequence shown in SEQID NO:2 under stringent conditions as more fully described below.

[0066] To determine the percent identity of two amino acid sequences orof two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, the length of a reference sequencealigned for comparison purposes is at least 30%, preferably at least40%, more preferably at least 50%, even more preferably at least 60%,and even more preferably at least 70%, 80%, or 90% of the length of thereference sequence (e.g., when aligning a second sequence to the aminoacid sequences herein having 502 amino acid residues, at least 165,preferably at least 200, more preferably at least 250, even morepreferably at least 300, and even more preferably at least 350, 400,450, and 500 amino acid residues are aligned). The amino acid residuesor nucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

[0067] The invention also encompasses polypeptides having a lower degreeof identity but having sufficient similarity so as to perform one ormore of the same functions performed by the carboxypeptidase. Similarityis determined by conserved amino acid substitution. Such substitutionsare those that substitute a given amino acid in a polypeptide by anotheramino acid of like characteristics. Conservative substitutions arelikely to be phenotypically silent. Typically seen as conservativesubstitutions are the replacements, one for another, among the aliphaticamino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residuesSer and Thr, exchange of the acidic residues Asp and Glu, substitutionbetween the amide residues Asn and Gln, exchange of the basic residuesLys and Arg and replacements among the aromatic residues Phe, Tyr.Guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990). TABLE 1 Conservative Amino Acid Substitutions. AromaticPhenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine ValinePolar Giutamine Asparagine Basic Arginine Lysine Histidine AcidicAspartic Acid Glutamic Acid Small Alamne Serine Threonine MethionineGlycine

[0068] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991).

[0069] A preferred, non-limiting example of such a mathematicalalgorithm is described in Karlin et al. (1993) Proc. Natl. Acad. Sci.USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs (version 2.0) as described in Altschul et al. (1997)Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,NBLAST) can be used. See www.ncbi.nlm.nih.gov. In one embodiment,parameters for sequence comparison can be set at score=100,wordlength=12, or can be varied (e.g., W=5 or W=20).

[0070] In a preferred embodiment, the percent identity between two aminoacid sequences is determined using the Needleman et al. (1970) (J. Mol.Biol. 48:444-453) algorithm which has been incorporated into the GAPprogram in the GCG software package (available at www.gcg.com), usingeither a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16,14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Inyet another preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (Devereux et al. (1984) Nucleic Acids Res. 12(1):387)(available at www.gcg.com), using a NWSgapdna. CMP matrix and a gapweight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or6.

[0071] Another preferred, non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller, CABIOS (1989). Such an algorithm is incorporated intothe ALIGN program (version 2.0) which is part of the CGC sequencealignment software package. When utilizing the ALIGN program forcomparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 can be used. Additionalalgorithms for sequence analysis are known in the art and includeADVANCE and ADAM as described in Torellis et al. (1994) Comput. Appl.Biosci. 10:3-5; and FASTA described in Pearson et al. (1988) PNAS85:2444-8.

[0072] A variant polypeptide can differ in amino acid sequence by one ormore substitutions, deletions, insertions, inversions, fusions, andtruncations or a combination of any of these.

[0073] Variant polypeptides can be fully functional or can lack functionin one or more activities. Thus, in the present case, variations canaffect the function, for example, of one or more of the regions relatingto peptide binding, specificity, or hydrolysis, regulatory/allostericregions, regions involved in membrane association, regions involved inmodification or activation of the carboxypeptidase, such asglycosylation, phosphorylation, and myristoylation, and any metalbinding regions.

[0074] Fully functional variants typically contain only conservativevariation or variation in non-critical residues or in non-criticalregions. Functional variants can also contain substitution of similaramino acids, which results in no change or an insignificant change infunction. Alternatively, such substitutions may positively or negativelyaffect function to some degree.

[0075] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0076] As indicated, variants can be naturally-occurring or can be madeby recombinant means or chemical synthesis to provide useful and novelcharacteristics for the carboxypeptidase polypeptide. This includespreventing immunogenicity from pharmaceutical formulations by preventingprotein aggregation.

[0077] Useful variations further include alteration of catalyticactivity. For example, one embodiment involves a variation at thebinding site that results in binding but not hydrolysis, or slowerhydrolysis, of substrate. A further useful variation at the same sitecan result in altered affinity for substrate. Useful variations alsoinclude changes that provide for affinity for another substrate. Anotheruseful variation includes one that prevents modification of thecarboxypeptidase. Another useful variation includes variation in theregion that provides for altered membrane association. Another usefulvariation provides a fusion protein in which one or more regions areoperationally fused to one or more regions from anothercarboxypeptidase.

[0078] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al. (1985) Science244:1081-1085). The latter procedure introduces single alanine mutationsat every residue in the molecule. The resulting mutant molecules arethen tested for biological activity, such as peptide hydrolysis in vitroor peptide-dependent in vitro activity, such as proliferative activity.Sites that are critical for binding can also be determined by structuralanalysis such as crystallization, nuclear magnetic resonance orphotoaffinity labeling (Smith et al. (1992) J. Mol. Biol. 224:899-904;de Vos et al. (1992) Science 255:306-312).

[0079] Substantial homology can be to the entire nucleic acid or aminoacid sequence or to fragments of these sequences.

[0080] The invention thus also includes polypeptide fragments of thecarboxypeptidase. Fragments can be derived from the amino acid sequenceshown in SEQ ID NO:1. However, the invention also encompasses fragmentsof the variants of the carboxypeptidase as described herein.

[0081] The fragments to which the invention pertains, however, are notto be construed as encompassing fragments that may be disclosed prior tothe present invention.

[0082] Accordingly, a fragment can comprise at least about 11, 12, 13,14, 15, 20, 25, 30, 35, 40, 45, 50 or more contiguous amino acids.Fragments can retain one or more of the biological activities of theprotein, for example the ability to bind to or hydrolyze peptides, aswell as fragments that can be used as an immunogen to generatecarboxypeptidase antibodies.

[0083] Biologically active fragments (peptides which are, for example,5, 7, 10, 12, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more aminoacids in length) can comprise a region or motif, e.g., regions relatingto peptide binding, specificity, or hydrolysis, regulatory/allostericregions, regions involved in membrane association, regions involved inmodification or activation of the carboxypeptidase, such asglycosylation, phosphorylation, and myristoylation, and any metalbinding regions.

[0084] Such regions or motifs can be identified by means of routinecomputerized homology searching procedures.

[0085] Fragments, for example, can extend in one or both directions fromthe functional site to encompass 5, 10, 15, 20, 30, 40, 50, or up to 100amino acids. Further, fragments can include sub-fragments of thespecific domains mentioned above, which sub-fragments retain thefunction of the region or domain from which they are derived.

[0086] These regions can be identified by well-known methods involvingcomputerized homology analysis.

[0087] The invention also provides fragments with immunogenicproperties. These contain an epitope-bearing portion of thecarboxypeptidase and variants. These epitope-bearing peptides are usefulto raise antibodies that bind specifically to a carboxypeptidasepolypeptide or region or fragment. These peptides can contain at least10, 12, at least 14, or between at least about 15 to about 30 aminoacids.

[0088] Non-limiting examples of antigenic polypeptides that can be usedto generate antibodies include but are not limited to peptides derivedfrom an extracellular region. Regions having a high antigenicity indexare shown in FIG. 3. However, intracellularly-made antibodies(“intrabodies”) are also encompassed, which would recognizeintracellular peptide regions.

[0089] The epitope-bearing carboxypeptidase polypeptides may be producedby any conventional means (Houghten, R. A. (1985) Proc. Natl. Acad. Sci.USA 82:5131-5135). Simultaneous multiple peptide synthesis is describedin U.S. Pat. No. 4,631,211.

[0090] Fragments can be discrete (not fused to other amino acids orpolypeptides) or can be within a larger polypeptide. Further, severalfragments can be comprised within a single larger polypeptide. In oneembodiment a fragment designed for expression in a host can haveheterologous pre- and pro-polypeptide regions fused to the aminoterminus of the carboxypeptidase fragment and an additional region fusedto the carboxyl terminus of the fragment.

[0091] The invention thus provides chimeric or fusion proteins. Thesecomprise a carboxypeptidase peptide sequence operatively linked to aheterologous peptide having an amino acid sequence not substantiallyhomologous to the carboxypeptidase. “Operatively linked” indicates thatthe carboxypeptidase peptide and the heterologous peptide are fusedin-frame. The heterologous peptide can be fused to the N-terminus orC-terminus of the carboxypeptidase or can be internally located.

[0092] In one embodiment the fusion protein does not affectcarboxypeptidase function per se. For example, the fusion protein can bea GST-fusion protein in which the carboxypeptidase sequences are fusedto the C-terminus of the GST sequences. Other types of fusion proteinsinclude, but are not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL4 fusions, poly-Hisfusions and Ig fusions. Such fusion proteins, particularly poly-Hisfusions, can facilitate the purification of recombinantcarboxypeptidase. In certain host cells (e.g., mammalian host cells),expression and/or secretion of a protein can be increased by using aheterologous signal sequence. Therefore, in another embodiment, thefusion protein contains a heterologous signal sequence at itsN-terminus.

[0093] EP-A-O 464 533 discloses fusion proteins comprising variousportions of immunoglobulin constant regions. The Fc is useful in therapyand diagnosis and thus results, for example, in improved pharmacokineticproperties (EP-A 0232 262). In drug discovery, for example, humanproteins have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists (Bennett et al.(1995) J. Mol. Recog. 8:52-58 (1995) and Johanson et al. J. Biol. Chem.270:9459-9471). Thus, this invention also encompasses soluble fusionproteins containing a carboxypeptidase polypeptide and various portionsof the constant regions of heavy or light chains of immunoglobulins ofvarious subclass (IgG, IgM, IgA, IgE). Preferred as immunoglobulin isthe constant part of the heavy chain of human IgG, particularly IgG1,where fusion takes place at the hinge region. For some uses it isdesirable to remove the Fc after the fusion protein has been used forits intended purpose, for example when the fusion protein is to be usedas antigen for immunizations. In a particular embodiment, the Fc partcan be removed in a simple way by a cleavage sequence, which is alsoincorporated and can be cleaved with factor Xa.

[0094] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al. (1992) Current Protocols in Molecular Biology). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A carboxypeptidase-encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the carboxypeptidase.

[0095] Another form of fusion protein is one that directly affectscarboxypeptidase functions. Accordingly, a carboxypeptidase polypeptideis encompassed by the present invention in which one or more of thecarboxypeptidase regions (or parts thereof) has been replaced byhomologous regions (or parts thereof) from another carboxypeptidase.Accordingly, various permutations are possible. For example, the activesite region, or subregion thereof, can be replaced with the active siteregion or subregion from another carboxypeptidase. As a further example,the membrane-associated region, or parts thereof, can be replaced. Thus,chimeric carboxypeptidases can be formed in which one or more of thenative regions has been replaced by another.

[0096] It is understood, however, that sites could be derived fromcarboxypeptidases that occur in the mammalian genome but which have notyet been discovered or characterized. Such sites include, but are notlimited to, regions relating to peptide binding, specificity, orhydrolysis, regulatory/allosteric regions, regions involved in membraneassociation, regions involved in modification or activation of thecarboxypeptidase, such as glycosylation, phosphorylation, andmyristoylation, and any metal binding regions, and any other functionalsite disclosed herein.

[0097] The isolated carboxypeptidase protein can be purified from cellsthat naturally express it, such as from those disclosed herein,especially purified from cells that have been altered to express it(recombinant), or synthesized using known protein synthesis methods.Preferred tissues in which the carboxypeptidase is expressed include,but are not limited to, prostate, breast, skeletal muscle, brain,testis, and thyroid. Cells also include colon, breast, and lungcarcinoma.

[0098] In one embodiment, the protein is produced by recombinant DNAtechniques. For example, a nucleic acid molecule encoding thecarboxypeptidase polypeptide is cloned into an expression vector, theexpression vector introduced into a host cell and the protein expressedin the host cell. The protein can then be isolated from the cells by anappropriate purification scheme using standard protein purificationtechniques. Polypeptides often contain amino acids other than the 20amino acids commonly referred to as the 20 naturally-occurring aminoacids. Further, many amino acids, including the terminal amino acids,may be modified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally inpolypeptides are described in basic texts, detailed monographs, and theresearch literature, and they are well known to those of skill in theart.

[0099] Accordingly, the polypeptides also encompass derivatives oranalogs in which a substituted amino acid residue is not one encoded bythe genetic code, in which a substituent group is included, in which themature polypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or in which the additional amino acids are fused to the maturepolypeptide, such as a leader or secretory sequence or a sequence forpurification of the mature polypeptide or a pro-protein sequence.

[0100] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphatidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0101] Such modifications are well-known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2nded., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (1990) Meth.Enzymol. 182: 626-646) and Rattan et al. (1992) Ann. N. Y. Acad. Sci.663:48-62).

[0102] As is also well known, polypeptides are not always entirelylinear. For instance, polypeptides may be branched as a result ofubiquitination, and they may be circular, with or without branching,generally as a result of post-translation events, including naturalprocessing events and events brought about by human manipulation whichdo not occur naturally. Circular, branched and branched circularpolypeptides may be synthesized by non-translational natural processesand by synthetic methods.

[0103] Modifications can occur anywhere in a polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. Blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally-occurring andsynthetic polypeptides. For instance, the aminoterminal residue ofpolypeptides made in E. coli, prior to proteolytic processing, ahnostinvariably will be N-formylmethionine.

[0104] The modifications can be a function of how the protein is made.For recombinant polypeptides, for example, the modifications will bedetermined by the host cell posttranslational modification capacity andthe modification signals in the polypeptide amino acid sequence.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcells often carry out the same posttranslational glycosylations asmammalian cells and, for this reason, insect cell expression systemshave been developed to efficiently express mammalian proteins havingnative patterns of glycosylation. Similar considerations apply to othermodifications.

[0105] The same type of modification may be present in the same orvarying degree at several sites in a given polypeptide. Also, a givenpolypeptide may contain more than one type of modification.

[0106] Polypeptide Uses

[0107] The protein sequences of the present invention can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul et al. (1990) J. Mol Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the proteins of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See www.ncbi.nlm.nih.gov.

[0108] The carboxypeptidase polypeptides are useful for producingantibodies specific for the carboxypeptidase, regions, or fragments.Regions having a high antigenicity index score are shown in FIG. 3.

[0109] The carboxypeptidase polypeptides are useful for biologicalassays related to carboxypeptidases. Such assays involve any of theknown carboxypeptidase functions or activities or properties useful fordiagnosis and treatment of carboxypeptidase-related conditions.

[0110] The carboxypeptidase polypeptides are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the carboxypeptidase,as a biopsy or expanded in cell culture. In one embodiment, however,cell-based assays involve recombinant host cells expressing thecarboxypeptidase.

[0111] Determining the ability of the test compound to interact with thecarboxypeptidase can also comprise determining the ability of the testcompound to preferentially bind to the polypeptide as compared to theability of a known binding molecule to bind to the polypeptide.

[0112] The polypeptides can be used to identify compounds that modulatecarboxypeptidase activity. Such compounds, for example, can increase ordecrease affinity or rate of binding to substrate, compete withsubstrate for binding to the carboxypeptidase, or displace substratebound to the carboxypeptidase. Both carboxypeptidase and appropriatevariants and fragments can be used in high-throughput screens to assaycandidate compounds for the ability to bind to the carboxypeptidase.These compounds can be further screened against a functionalcarboxypeptidase to determine the effect of the compound on thecarboxypeptidase activity. Compounds can be identified that activate(agonist) or inactivate (antagonist) the carboxypeptidase to a desireddegree. Modulatory methods can be performed in vitro (e.g., by culturingthe cell with the agent) or, alternatively, in vivo (e.g., byadministering the agent to a subject.

[0113] The carboxypeptidase polypeptides can be used to screen acompound for the ability to stimulate or inhibit interaction between thecarboxypeptidase protein and a target molecule that normally interactswith the carboxypeptidase protein. The assay includes the steps ofcombining the carboxypeptidase protein with a candidate compound underconditions that allow the carboxypeptidase protein or fragment tointeract with the target molecule, and to detect the formation of acomplex between the carboxypeptidase protein and the target or to detectthe biochemical consequence of the interaction with the carboxypeptidaseand the target, such as any of the associated effects of peptidehydrolysis.

[0114] Determining the ability of the carboxypeptidase to bind to atarget molecule can also be accomplished using a technology such asreal-time Bimolecular Interaction Analysis (BIA). Sjolander et al.(1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin.Struct. Biol. 5:699-705. As used herein, “BIA” is a technology forstudying biospecific interactions in real time, without labeling any ofthe interactants (e.g., BIAcore™). Changes in the optical phenomenonsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

[0115] The test compounds of the present invention can be obtained usingany of the numerous approaches in combinatorial library methods known inthe art, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to polypeptide libraries, whilethe other four approaches are applicable to polypeptide, non-peptideoligomer or small molecule libraries of compounds (Lam, K. S. (1997)Anticancer Drug Des. 12:145).

[0116] Examples of methods for the synthesis of molecular libraries canbe found in the art, for example in DeWitt et al. (1993) Proc. Natl.Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233. Libraries ofcompounds may be presented in solution (e.g., Houghten (1992)Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84),chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No.5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al.(1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott andSmith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406);(Cwirla et al. (1990) Proc. Natl. Acad. Sci. 97:6378-6382); (Felici(1991) J. Mol. Biol. 222:301-310); (Ladner supra).

[0117] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al. (1991) Nature 354:82-84;Houghten et al. (1991) Nature 354:84-86) and combinatorialchemistry-derived molecular libraries made of D- and/or L- configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal. (1993) Cell 72:767-778); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0118] One candidate compound is a soluble full-length carboxypeptidaseor fragment that competes for substrate binding. Other candidatecompounds include mutant carboxypeptidases or appropriate fragmentscontaining mutations that affect carboxypeptidase function and thuscompete for substrate. Accordingly, a fragment that competes forsubstrate, for example with a higher affinity, or a fragment that bindsthe substrate but does not degrade it, is encompassed by the invention.

[0119] The invention provides other end points to identify compoundsthat modulate (stimulate or inhibit) carboxypeptidase activity. Theassays typically involve an assay of cellular events that indicatecarboxypeptidase activity. Thus, the expression of genes that are up- ordown-regulated in response to carboxypeptidase activity can be assayed.In one embodiment, the regulatory region of such genes can be operablylinked to a marker that is easily detectable, such as luciferase.Alternatively, modification of the carboxypeptidase could also bemeasured.

[0120] Any of the biological or biochemical functions mediated by thecarboxypeptidase can be used as an endpoint assay. These include all ofthe biochemical or biochemical/biological events described herein, inthe references cited herein, incorporated by reference for theseendpoint assay targets, and other functions known to those of ordinaryskill in the art.

[0121] In the case of the carboxypeptidase, assays for specific endpoints can include assays for peptide hydrolysis (e.g., amino acidproduction or change in substrate peptide size).

[0122] Binding and/or activating compounds can also be screened by usingchimeric carboxypeptidase proteins in which one or more regions/domains,segments, sites, and the like, as disclosed herein, or parts thereof,can be replaced by their heterologous counterparts derived from othercarboxypeptidases. For example, a catalytic region can be used thatinteracts with a different specificity and/or affinity than the nativecarboxypeptidase. Accordingly, a different set of cellular components isavailable as an end-point assay for activation. Alternatively, amembrane-associated portion or subregions can be replaced with themembrane portion or subregions specific to a host cell that is differentfrom the host cell from which the native carboxypeptidase is derived.This allows for assays to be performed in other than the specific hostcell from which the carboxypeptidase is derived. Alternatively, aheterologous substrate recognition sequence can replace the nativesequence. This can result in having an effect on a different cellularpathway. Accordingly, a different set of cellular components isavailable as an endpoint assay for activation. Activation can also bedetected by a reporter gene containing an easily detectable codingregion operably linked to a transcriptional regulatory sequence that ispart of the native pathway.

[0123] The carboxypeptidase polypeptides are also useful in competitionbinding assays in methods designed to discover compounds that interactwith the carboxypeptidase. Thus, a compound is exposed to acarboxypeptidase polypeptide under conditions that allow the compound tobind or to otherwise interact with the polypeptide. Solublecarboxypeptidase polypeptide is also added to the mixture. If the testcompound interacts with the soluble carboxypeptidase polypeptide, itdecreases the amount of complex formed or activity from thecarboxypeptidase target. This type of assay is particularly useful incases in which compounds are sought that interact with specific regionsof the carboxypeptidase. Thus, the soluble polypeptide that competeswith the target carboxypeptidase region is designed to contain peptidesequences corresponding to the region of interest.

[0124] Another type of competition-binding assay can be used to discovercompounds that interact with specific functional sites. As an example,peptide substrate and a candidate compound can be added to a sample ofthe carboxypeptidase. Compounds that interact with the carboxypeptidaseat the same site as the peptide will reduce the amount of complex formedbetween the carboxypeptidase and the peptide. Accordingly, it ispossible to discover a compound that specifically prevents interactionbetween the carboxypeptidase and the peptide. Another example involvesadding a candidate compound to a sample of carboxypeptidase and peptidesubstrate. A compound that competes with the peptide substrate willreduce the amount of hydrolysis or binding of the substrate to thecarboxypeptidase. Accordingly, compounds can be discovered that directlyinteract with the carboxypeptidase and compete with the substrate. Suchassays can involve any other component that interacts with thecarboxypeptidase.

[0125] To perform cell free drug screening assays, it is desirable toimmobilize either the carboxypeptidase, or fragment, or its targetmolecule to facilitate separation of complexes from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay.

[0126] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase/carboxypeptidase fusionproteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtitre plates,which are then combined with the cell lysates (e.g., ³⁵S-labeled) andthe candidate compound, and the mixture incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads are washed to remove anyunbound label, and the matrix immobilized and radiolabel determineddirectly, or in the supernatant after the complexes is dissociated.Alternatively, the complexes can be dissociated from the matrix,separated by SDS-PAGE, and the level of carboxypeptidase-binding proteinfound in the bead fraction quantitated from the gel using standardelectrophoretic techniques. For example, either the polypeptide or itstarget molecule can be immobilized utilizing conjugation of biotin andstreptavidin using techniques well known in the art. Alternatively,antibodies reactive with the protein but which do not interfere withbinding of the protein to its target molecule can be derivatized to thewells of the plate, and the protein trapped in the wells by antibodyconjugation. Preparations of a carboxypeptidase-binding targetcomponent, such as peptide substrate, and a candidate compound areincubated in the carboxypeptidase-presenting wells and the amount ofcomplex trapped in the well can be quantitated. Methods for detectingsuch complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the carboxypeptidase target molecule, or whichare reactive with carboxypeptidase and compete with the target molecule;as well as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the target molecule.

[0127] Modulators of carboxypeptidase activity identified according tothese drug screening assays can be used to treat a subject with adisorder related to the carboxypeptidase, by treating cells that expressthe carboxypeptidase, such as those disclosed here. Preferred tissues inwhich the carboxypeptidase is expressed include, but are not limited to,prostate, breast, skeletal muscle, brain, testes, thyroid, and fetalliver, kidney, and heart. Further, preferred tissues include, but arenot limited to, colon and breast carcinoma, and lung carcinoma,especially squamous cell carcinoma. In addition, this carboxypeptidaseis overexpressed in breast, lung and colon tumors as disclosed herein.Accordingly, expression of the carboxypeptidase is especially relevantto cancer treatment, including invasion and metastasis. These methods oftreatment include the steps of administering the modulators ofcarboxypeptidase activity in a pharmaceutical composition as describedherein, to a subject in need of such treatment.

[0128] Further disorders in which the carboxypeptidase expression isrelevant include, but are not limited to, any disorders involving thecells in which the carboxypeptidase is expressed as disclosed herein.

[0129] Disorders involving the lung include, but are not limited to,congenital anomalies; atelectasis; diseases of vascular origin, such aspulmonary congestion and edema, including hemodynamic pulmonary edemaand edema caused by microvascular injury, adult respiratory distresssyndrome (diffuse alveolar damage), pulmonary embolism, hemorrhage, andinfarction, and pulmonary hypertension and vascular sclerosis; chronicobstructive pulmonary disease, such as emphysema, chronic bronchitis,bronchial asthma, and bronchiectasis; diffuse interstitial(infiltrative, restrictive) diseases, such as pneumoconioses,sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitialpneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia(pulmonary infiltration with eosinophilia), Bronchiolitisobliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes,including Goodpasture syndrome, idiopathic pulmonary hemosiderosis andother hemorrhagic syndromes, pulmonary involvement in collagen vasculardisorders, and pulmonary alveolar proteinosis; complications oftherapies, such as drug-induced lung disease, radiation-induced lungdisease, and lung transplantation; tumors, such as bronchogeniccarcinoma, including paraneoplastic syndromes, bronchioloalveolarcarcinoma, neuroendocrine tumors, such as bronchial carcinoid,miscellaneous tumors, and metastatic tumors; pathologies of the pleura,including inflammatory pleural effusions, noninflammatory pleuraleffusions, pneumothorax, and pleural tumors, including solitary fibroustumors (pleural fibroma) and malignant mesothelioma.

[0130] Disorders involving the colon include, but are not limited to,congenital anomalies, such as atresia and stenosis, Meckel diverticulum,congenital aganglionic megacolon-Hirschsprung disease; enterocolitis,such as diarrhea and dysentery, infectious enterocolitis, includingviral gastroenteritis, bacterial enterocolitis, necrotizingenterocolitis, antibiotic-associated colitis (pseudomembranous colitis),and collagenous and lymphocytic colitis, miscellaneous intestinalinflammatory disorders, including parasites and protozoa, acquiredimmunodeficiency syndrome, transplantation, drug-induced intestinalinjury, radiation enterocolitis, neutropenic colitis (typhlitis), anddiversion colitis; idiopathic inflammatory bowel disease, such as Crohndisease and ulcerative colitis; tumors of the colon, such asnon-neoplastic polyps, adenomas, familial syndromes, colorectalcarcinogenesis, colorectal carcinoma, and carcinoid tumors.

[0131] Disorders involving the liver include, but are not limited to,hepatic injury; jaundice and cholestasis, such as bilirubin and bileformation; hepatic failure and cirrhosis, such as cirrhosis, portalhypertension, including ascites, portosystemic shunts, and splenomegaly;infectious disorders, such as viral hepatitis, including hepatitis A-Einfection and infection by other hepatitis viruses, clinicopathologicsyndromes, such as the carrier state, asymptomatic infection, acuteviral hepatitis, chronic viral hepatitis, and fulminant hepatitis;autoimmune hepatitis; drug- and toxin-induced liver disease, such asalcoholic liver disease; inborn errors of metabolism and pediatric liverdisease, such as hemochromatosis, Wilson disease, a₁-antitrypsindeficiency, and neonatal hepatitis; intrahepatic biliary tract disease,such as secondary biliary cirrhosis, primary biliary cirrhosis, primarysclerosing cholangitis, and anomalies of the biliary tree; circulatorydisorders, such as impaired blood flow into the liver, including hepaticartery compromise and portal vein obstruction and thrombosis, impairedblood flow through the liver, including passive congestion andcentrilobular necrosis and peliosis hepatis, hepatic vein outflowobstruction, including hepatic vein thrombosis (Budd-Chiari syndrome)and veno-occlusive disease; hepatic disease associated with pregnancy,such as preeclampsia and eclampsia, acute fatty liver of pregnancy, andintrehepatic cholestasis of pregnancy; hepatic complications of organ orbone marrow transplantation, such as drug toxicity after bone marrowtransplantation, graft-versus-host disease and liver rejection, andnonimmunologic damage to liver allografts; tumors and tumorousconditions, such as nodular hyperplasias, adenomas, and malignanttumors, including primary carcinoma of the liver and metastatic tumors.

[0132] Disorders involving the brain include, but are limited to,disorders involving neurons, and disorders involving glia, such asastrocytes, oligodendrocytes, ependyrnal cells, and microglia; cerebraledema, raised intracranial pressure and herniation, and hydrocephalus;malformations and developmental diseases, such as neural tube defects,forebrain anomalies, posterior fossa anomalies, and syringomyelia andhydromyelia; perinatal brain injury; cerebrovascular diseases, such asthose related to hypoxia, ischemia, and infarction, includinghypotension, hypoperfusion, and low-flow states—global cerebral ischemiaand focal cerebral ischemia—infarction from obstruction of local bloodsupply, intracranial hemorrhage, including intracerebral(intraparenchymal) hemorrhage, subarachnoid hemorrhage and rupturedberry aneurysms, and vascular malformations, hypertensivecerebrovascular disease, including lacunar infarcts, slit hemorrhages,and hypertensive encephalopathy; infections, such as acute meningitis,including acute pyogenic (bacterial) meningitis and acute aseptic(viral) meningitis, acute focal suppurative infections, including brainabscess, subdural empyema, and extradural abscess, chronic bacterialmeningoencephalitis, including tuberculosis and mycobacterioses,neurosyphilis, and neuroborreliosis (Lyme disease), viralmeningoencephalitis, including arthropod-bome (Arbo) viral encephalitis,Herpes simplex virus Type 1, Herpes simplex virus Type 2,Varicalla-zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis,rabies, and human immunodeficiency virus 1, including HIV-1meningoencephalitis (subacute encephalitis), vacuolar myelopathy,AIDS-associated myopathy, peripheral neuropathy, and AIDS in children,progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, fungal meningoencephalitis, other infectious diseasesof the nervous system; transmissible spongiform encephalopathies (priondiseases); demyelinating diseases, including multiple sclerosis,multiple sclerosis variants, acute disseminated encephalomyelitis andacute necrotizing hemorrhagic encephalomyelitis, and other diseases withdemyelination; degenerative diseases, such as degenerative diseasesaffecting the cerebral cortex, including Alzheimer disease and Pickdisease, degenerative diseases of basal ganglia and brain stem,including Parkinsonism, idiopathic Parkinson disease (paralysisagitans), progressive supranuclear palsy, corticobasal degenration,multiple system atrophy, including striatonigral degenration, Shy-Dragersyndrome, and olivopontocerebellar atrophy, and Huntington disease;spinocerebellar degenerations, including spinocerebellar ataxias,including Friedreich ataxia, and ataxia-telanglectasia, degenerativediseases affecting motor neurons, including amyotrophic lateralsclerosis (motor neuron disease), bulbospinal atrophy (Kennedysyndrome), and spinal muscular atrophy; inborn errors of metabolism,such as leukodystrophies, including Krabbe disease, metachromaticleukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, andCanavan disease, mitochondrial encephalomyopathies, including Leighdisease and other mitochondrial encephalomyopathies; toxic and acquiredmetabolic diseases, including vitamin deficiencies such as thiamine(vitamin B₁) deficiency and vitamin B₁₂ deficiency, neurologic sequelaeof metabolic disturbances, including hypoglycemia, hyperglycemia, andhepatic encephatopathy, toxic disorders, including carbon monoxide,methanol, ethanol, and radiation, including combined methotrexate andradiation-induced injury; tumors, such as gliomas, includingastrocytoma, including fibrillary (diffuse) astrocytoma and glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, andbrain stem glioma, oligodendroglioma, and ependymoma and relatedparaventricular mass lesions, neuronal tumors, poorly differentiatedneoplasms, including medulloblastoma, other parenchymal tumors,including primary brain lymphoma, germ cell tumors, and pinealparenchymal tumors, meningiomas, metastatic tumors, paraneoplasticsyndromes, peripheral nerve sheath tumors, including schwannoma,neurofibroma, and malignant peripheral nerve sheath tumor (malignantschwannoma), and neurocutaneous syndromes (phakomatoses), includingneurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindaudisease.

[0133] Disorders involving the heart, include but are not limited to,heart failure, including but not limited to, cardiac hypertrophy,left-sided heart failure, and right-sided heart failure; ischemic heartdisease, including but not limited to angina pectoris, myocardialinfarction, chronic ischemic heart disease, and sudden cardiac death;hypertensive heart disease, including but not limited to, systemic(left-sided) hypertensive heart disease and pulmonary (right-sided)hypertensive heart disease; valvular heart disease, including but notlimited to, valvular degeneration caused by calcification, such ascalcific aortic stenosis, calcification of a congenitally bicuspidaortic valve, and mitral annular calcification, and myxomatousdegeneration of the mitral valve (mitral valve prolapse), rheumaticfever and rheumatic heart disease, infective endocarditis, andnoninfected vegetations, such as nonbacterial thrombotic endocarditisand endocarditis of systemic lupus erythematosus (Libman-Sacks disease),carcinoid heart disease, and complications of artificial valves;myocardial disease, including but not limited to dilated cardiomyopathy,hypertrophic cardiomyopathy, restrictive cardiomyopathy, andmyocarditis; pericardial disease, including but not limited to,pericardial effusion and hemopericardium and pericarditis, includingacute pericarditis and healed pericarditis, and rheumatoid heartdisease; neoplastic heart disease, including but not limited to, primarycardiac tumors, such as myxoma, lipoma, papillary fibroelastoma,rhabdomyoma, and sarcoma, and cardiac effects of noncardiac neoplasms;congenital heart disease, including but not limited to, left-to-rightshunts—late cyanosis, such as atrial septal defect, ventricular septaldefect, patent ductus arteriosus, and atrioventricular septal defect,right-to-left shunts—early cyanosis, such as tetralogy of fallot,transposition of great arteries, truncus arteriosus, tricuspid atresia,and total anomalous pulmonary venous connection, obstructive congenitalanomalies, such as coarctation of aorta, pulmonary stenosis and atresia,and aortic stenosis and atresia, and disorders involving cardiactransplantation.

[0134] The carboxypeptidase polypeptides are thus useful for treating acarboxypeptidase-associated disorder characterized by aberrantexpression or activity of a carboxypeptidase. In one embodiment, themethod involves administering an agent (e.g., an agent identified by ascreening assay described herein), or combination of agents thatmodulates (e.g., upregulates or downregulates) expression or activity ofthe protein. In another embodiment, the method involves administeringthe carboxypeptidase as therapy to compensate for reduced or aberrantexpression or activity of the protein.

[0135] Methods for treatment include but are not limited to the use ofsoluble carboxypeptidase or fragments of the carboxypeptidase proteinthat compete for substrate. These carboxypeptidases or fragments canhave a higher affinity for the target so as to provide effectivecompetition.

[0136] Stimulation of activity is desirable in situations in which theprotein is abnormally downregulated and/or in which increased activityis likely to have a beneficial effect. Likewise, inhibition of activityis desirable in situations in which the protein is abnormallyupregulated and/or in which decreased activity is likely to have abeneficial effect. In one example of such a situation, a subject has adisorder characterized by aberrant development or cellulardifferentiation. In another example, the subject has a proliferativedisease (e.g., cancer) or a disorder characterized by an aberranthematopoietic response. In another example, it is desirable to achievetissue regeneration in a subject (e.g., where a subject has undergonebrain or spinal cord injury and it is desirable to regenerate neuronaltissue in a regulated manner).

[0137] In yet another aspect of the invention, the proteins of theinvention can be used as “bait proteins” in a two-hybrid assay orthree-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al.(1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchiet al. (1993) Oncogene 8:1693-1696; and Brent WO 94/10300), to identifyother proteins (captured proteins) which bind to or interact with theproteins of the invention and modulate their activity.

[0138] The carboxypeptidase polypeptides also are useful to provide atarget for diagnosing a disease or predisposition to disease related tothe carboxypeptidase, including, but not limited to, those diseasesdiscussed herein, and particularly breast, colon and lung carcinoma.Targets are useful for diagnosing a disease or predisposition to diseasemediated by the carboxypeptidase, especially in the tissues shown inthose found herein, especially in prostate, breast, skeletal muscle,brain, testis, thyroid, and carcinomas, such as in colon, breast, andlung. Accordingly, methods are provided for detecting the presence, orlevels of, the carboxypeptidase in a cell, tissue, or organism. Themethod involves contacting a biological sample with a compound capableof interacting with the carboxypeptidase such that the interaction canbe detected.

[0139] One agent for detecting carboxypeptidase is an antibody capableof selectively binding to carboxypeptidase. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0140] The carboxypeptidase also provides a target for diagnosing activedisease, or predisposition to disease, in a patient having a variantcarboxypeptidase. Thus, carboxypeptidase can be isolated from abiological sample and assayed for the presence of a genetic mutationthat results in an aberrant protein. This includes amino acidsubstitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered carboxypeptidase activity incell-based or cell-free assay, alteration in substrate binding ordegradation, antibody-binding pattern, altered isoelectric point, directamino acid sequencing, and any other of the known assay techniquesuseful for detecting mutations in a protein in general or in acarboxypeptidase specifically.

[0141] In vitro techniques for detection of carboxypeptidase includeenzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. Alternatively, the proteincan be detected in vivo in a subject by introducing into the subject alabeled anti-carboxypeptidase antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques. Particularlyuseful are methods, which detect the allelic variant of thecarboxypeptidase expressed in a subject, and methods, which detectfragments of the carboxypeptidase in a sample.

[0142] The carboxypeptidase polypeptides are also useful inpharmacogenomic analysis. Pharmacogenomics deal with clinicallysignificant hereditary variations in the response to drugs due toaltered drug disposition and abnormal action in affected persons. See,e.g., Eichelbaum, M. (1996) Clin. Exp. Pharmacol. Physiol.23(10-11):983-985, and Linder, M. W. (1997) Clin. Chem. 43(2):254-266.The clinical outcomes of these variations result in severe toxicity oftherapeutic drugs in certain individuals or therapeutic failure of drugsin certain individuals as a result of individual variation inmetabolism. Thus, the genotype of the individual can determine the way atherapeutic compound acts on the body or the way the body metabolizesthe compound. Further, the activity of drug metabolizing enzymes affectsboth the intensity and duration of drug action. Thus, thepharmacogenomics of the individual permit the selection of effectivecompounds and effective dosages of such compounds for prophylactic ortherapeutic treatment based on the individual's genotype. The discoveryof genetic polymorphisms in some drug metabolizing enzymes has explainedwhy some patients do not obtain the expected drug effects, show anexaggerated drug effect, or experience serious toxicity from standarddrug dosages. Polymorphisms can be expressed in the phenotype of theextensive metabolizer and the phenotype of the poor metabolizer.Accordingly, genetic polymorphism may lead to allelic protein variantsof the carboxypeptidase in which one or more of the carboxypeptidasefunctions in one population is different from those in anotherpopulation. The polypeptides thus allow a target to ascertain a geneticpredisposition that can affect treatment modality. Thus, in apeptide-based treatment, polymorphism may give rise to catalytic regionsthat are more or less active. Accordingly, dosage would necessarily bemodified to maximize the therapeutic effect within a given populationcontaining the polymorphism. As an alternative to genotyping, specificpolymorphic polypeptides could be identified.

[0143] The carboxypeptidase polypeptides are also useful for monitoringtherapeutic effects during clinical trials and other treatment. Thus,the therapeutic effectiveness of an agent that is designed to increaseor decrease gene expression, protein levels or carboxypeptidase activitycan be monitored over the course of treatment using the carboxypeptidasepolypeptides as an end-point target. The monitoring can be, for example,as follows: (i) obtaining a pre-administration sample from a subjectprior to administration of the agent; (ii) detecting the level ofexpression or activity of the protein in the pre-administration sample;(iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression or activity of theprotein in the post-administration samples; (v) comparing the level ofexpression or activity of the protein in the pre-administration samplewith the protein in the post-administration sample or samples; and (vi)increasing or decreasing the administration of the agent to the subjectaccordingly.

[0144] Antibodies

[0145] The invention also provides antibodies that selectively bind tothe carboxypeptidase and its variants and fragments. An antibody isconsidered to selectively bind, even if it also binds to other proteinsthat are not substantially homologous with the carboxypeptidase. Theseother proteins share homology with a fragment or domain of thecarboxypeptidase. This conservation in specific regions gives rise toantibodies that bind to both proteins by virtue of the homologoussequence. In this case, it would be understood that antibody binding tothe carboxypeptidase is still selective.

[0146] To generate antibodies, an isolated carboxypeptidase polypeptideis used as an immunogen to generate antibodies using standard techniquesfor polyclonal and monoclonal antibody preparation. Either thefull-length protein or antigenic peptide fragment can be used. Regionshaving a high antigenicity index are shown in FIG. 3.

[0147] Antibodies are preferably prepared from these regions or fromdiscrete fragments in these regions. However, antibodies can be preparedfrom any region of the peptide as described herein. A preferred fragmentproduces an antibody that diminishes or completely prevents substratehydrolysis or binding. Antibodies can be developed against the entirecarboxypeptidase or regions/domains of the carboxypeptidase as describedherein. Antibodies can also be developed against specific functionalsites as disclosed herein.

[0148] The antigenic peptide can comprise a contiguous sequence of atleast 12, 14, 15, or 30 amino acid residues. In one embodiment,fragments correspond to regions that are located on the surface of theprotein, e.g., hydrophilic regions. These fragments are not to beconstrued, however, as encompassing any fragments, which may bedisclosed prior to the invention.

[0149] Antibodies can be polyclonal or monoclonal. An intact antibody,or a fragment thereof (e.g. Fab or F(ab′)₂) can be used.

[0150] Detection can be facilitated by coupling (i.e., physicallylinking) the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

[0151] An appropriate immunogenic preparation can be derived fromnative, recombinantly expressed, or chemically synthesized peptides.

[0152] Antibody Uses

[0153] The antibodies can be used to isolate a carboxypeptidase bystandard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural carboxypeptidase from cells and recombinantly producedcarboxypeptidase expressed in host cells.

[0154] The antibodies are useful to detect the presence ofcarboxypeptidase in cells or tissues to determine the pattern ofexpression of the carboxypeptidase among various tissues in an organismand over the course of normal development.

[0155] The antibodies can be used to detect carboxypeptidase in situ, invitro, or in a cell lysate or supernatant in order to evaluate theabundance and pattern of expression.

[0156] The antibodies can be used to assess abnormal tissue distributionor abnormal expression during development.

[0157] Antibody detection of circulating fragments of the full lengthcarboxypeptidase can be used to identify carboxypeptidase turnover.

[0158] Further, the antibodies can be used to assess carboxypeptidaseexpression in disease states such as in active stages of the disease orin an individual with a predisposition toward disease related tocarboxypeptidase function. When a disorder is caused by an inappropriatetissue distribution, developmental expression, or level of expression ofthe carboxypeptidase protein, the antibody can be prepared against thenormal carboxypeptidase protein. If a disorder is characterized by aspecific mutation in the carboxypeptidase, antibodies specific for thismutant protein can be used to assay for the presence of the specificmutant carboxypeptidase. However, intracellularly-made antibodies(“intrabodies”) are also encompassed, which would recognizeintracellular carboxypeptidase peptide regions.

[0159] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Antibodies can be developed against the whole carboxypeptidase orportions of the carboxypeptidase, for example, the catalytic region.

[0160] The diagnostic uses can be applied, not only in genetic testing,but also in monitoring a treatment modality. Accordingly, wheretreatment is ultimately aimed at correcting carboxypeptidase expressionlevel or the presence of aberrant carboxypeptidases and aberrant tissuedistribution or developmental expression, antibodies directed againstthe carboxypeptidase or relevant fragments can be used to monitortherapeutic efficacy.

[0161] Antibodies accordingly can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure, e.g.,to, for example, determine the efficacy of a given treatment regimen.

[0162] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic carboxypeptidase can beused to identify individuals that require modified treatment modalities.

[0163] The antibodies are also useful as diagnostic tools as animmunological marker for aberrant carboxypeptidase analyzed byelectrophoretic mobility, isoelectric point, tryptic peptide digest, andother physical assays known to those in the art.

[0164] The antibodies are also useful for tissue typing. Thus, where aspecific carboxypeptidase has been correlated with expression in aspecific tissue, antibodies that are specific for this carboxypeptidasecan be used to identify a tissue type.

[0165] The antibodies are also useful in forensic identification.Accordingly, where an individual has been correlated with a specificgenetic polymorphism resulting in a specific polymorphic protein, anantibody specific for the polymorphic protein can be used as an aid inidentification.

[0166] The antibodies are also useful for inhibiting carboxypeptidasefunction, for example, blocking the catalytic site.

[0167] These uses can also be applied in a therapeutic context in whichtreatment involves inhibiting carboxypeptidase function. An antibody canbe used, for example, to block peptide substrate binding. Antibodies canbe prepared against specific fragments containing sites required forfunction or against intact carboxypeptidase associated with a cell.

[0168] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. For an overview of thistechnology for producing human antibodies, see Lonberg et al. (1995)Int. Rev. Immunol. 13:65-93. For a detailed discussion of thistechnology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, e.g., U.S. Pat.No. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806.

[0169] The invention also encompasses kits for using antibodies todetect the presence of a carboxypeptidase protein in a biologicalsample. The kit can comprise antibodies such as a labeled or labelableantibody and a compound or agent for detecting carboxypeptidase in abiological sample; means for determining the amount of carboxypeptidasein the sample; and means for comparing the amount of carboxypeptidase inthe sample with a standard. The compound or agent can be packaged in asuitable container. The kit can further comprise instructions for usingthe kit to detect carboxypeptidase.

[0170] Polynucleotides

[0171] The nucleotide sequences in SEQ ID NO:2 were obtained bysequencing the deposited human cDNA. Accordingly, the sequence of thedeposited clone is controlling as to any discrepancies between the twoand any reference to the sequence of SEQ ID NO:2 includes reference tothe sequence of the deposited cDNA.

[0172] The specifically disclosed cDNA comprises the coding region and5′ and 3′ untranslated sequences in SEQ ID NO:2.

[0173] The nucleic acid is expressed in those tissues as disclosedherein. In particular, the carboxypeptidase is expressed in prostate,breast, skeletal muscle, brain, testis, thyroid, and carcinomas, such asin colon, breast, and lung. In addition, this carboxypeptidase isoverexpressed in a number of breast, lung and colon tumor lines asdisclosed herein.

[0174] The invention provides isolated polynucleotides encoding thenovel carboxypeptidases. The term “carboxypeptidase polynucleotide” or“carboxypeptidase nucleic acid” refers to the sequence shown in SEQ IDNO:2 or in the deposited cDNA. The term “carboxypeptidasepolynucleotide” or “carboxypeptidase nucleic acid” further includesvariants and fragments of the carboxypeptidase polynucleotides.

[0175] An “isolated” carboxypeptidase nucleic acid is one that isseparated from other nucleic acid present in the natural source of thecarboxypeptidase nucleic acid. Preferably, an “isolated” nucleic acid isfree of sequences which naturally flank the carboxypeptidase nucleicacid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. However, there can be some flanking nucleotide sequences, forexample up to about 5KB. The important point is that thecarboxypeptidase nucleic acid is isolated from flanking sequences suchthat it can be subjected to the specific manipulations described herein,such as recombinant expression, preparation of probes and primers, andother uses specific to the carboxypeptidase nucleic acid sequences.

[0176] Moreover, an “isolated” nucleic acid molecule, such as a cDNA orRNA molecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

[0177] In some instances, the isolated material will form part of acomposition (for example, a crude extract containing other substances),buffer system or reagent mix. In other circumstances, the material maybe purified to essential homogeneity, for example as determined by PAGEor column chromatography such as HPLC. Preferably, an isolated nucleicacid comprises at least about 50, 80 or 90% (on a molar basis) of allmacromolecular species present.

[0178] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0179] In some instances, the isolated material will form part of acomposition (or example, a crude extract containing other substances),buffer system or reagent mix. In other circumstances, the material maybe purified to essential homogeneity, for example as determined by PAGEor column chromatography such as HPLC. Preferably, an isolated nucleicacid comprises at least about 50, 80 or 90% (on a molar basis) of allmacromolecular species present.

[0180] The carboxypeptidase polynucleotides can encode the matureprotein plus additional amino or carboxyterminal amino acids, or aminoacids interior to the mature polypeptide (when the mature form has morethan one polypeptide chain, for instance). Such sequences may play arole in processing of a protein from precursor to a mature form,facilitate protein trafficking, prolong or shorten protein half-life orfacilitate manipulation of a protein for assay or production, amongother things. As generally is the case in situ, the additional aminoacids may be processed away from the mature protein by cellular enzymes.

[0181] The carboxypeptidase polynucleotides include, but are not limitedto, the sequence encoding the mature polypeptide alone, the sequenceencoding the mature polypeptide and additional coding sequences, such asa leader or secretory sequence (e.g., a pre-pro or pro-proteinsequence), the sequence encoding the mature polypeptide, with or withoutthe additional coding sequences, plus additional non-coding sequences,for example introns and non-coding 5′ and 3′ sequences such astranscribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thepolynucleotide may be fused to a marker sequence encoding, for example,a peptide that facilitates purification.

[0182] Carboxypeptidase polynucleotides can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0183] Carboxypeptidase nucleic acid can comprise the nucleotidesequences shown in SEQ ID NO:2, corresponding to human osteoblast orbrain cDNA.

[0184] In one embodiment, the carboxypeptidase nucleic acid comprisesonly the coding region.

[0185] The invention further provides variant carboxypeptidasepolynucleotides, and fragments thereof, that differ from the nucleotidesequence shown in SEQ ID NO:2 due to degeneracy of the genetic code andthus encode the same protein as that encoded by the nucleotide sequenceshown in SEQ ID NO:2.

[0186] The invention also provides carboxypeptidase nucleic acidmolecules encoding the variant polypeptides described herein. Suchpolynucleotides may be naturally occurring, such as allelic variants(same locus), homologs (different locus), and orthologs (differentorganism), or may be constructed by recombinant DNA methods or bychemical synthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to polynucleotides,cells, or organisms. Accordingly, as discussed above, the variants cancontain nucleotide substitutions, deletions, inversions and insertions.

[0187] Typically, variants have a substantial identity with a nucleicacid molecules of SEQ ID NO:2 and the complements thereof Variation canoccur in either or both the coding and non-coding regions. Thevariations can produce both conservative and non-conservative amino acidsubstitutions.

[0188] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. These variants comprise a nucleotidesequence encoding a carboxypeptidase that is typically at least about50-55%, 55-60%, 60-65%, 65-70%, 70-75%, more typically at least about80-85%, and most typically at least about 90-95% or more homologous tothe nucleotide sequence shown in SEQ ID NO:2 or a fragment of thissequence. Such nucleic acid molecules can readily be identified as beingable to hybridize under stringent conditions, to the nucleotide sequenceshown in SEQ ID NO:2 or a fragment of the sequence. It is understoodthat stringent hybridization does not indicate substantial homologywhere it is due to general homology, such as poly A sequences, orsequences common to all or most proteins, all carboxypeptidases, allprolyl carboxypeptidases or all seryl carboxypeptidases. Moreover, it isunderstood that variants do not include any of the nucleic acidsequences that may have been disclosed prior to the invention.

[0189] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a polypeptide at least about 50-55%homologous to each other typically remain hybridized to each other. Theconditions can be such that sequences at least about 60-65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 90%,at least about 95% or more identical to each other remain hybridized toone another. Such stringent conditions are known to those skilled in theart and can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6, incorporated by reference. Oneexample of stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2× SSC, 0.1% SDS at 50-65° C. In another non-limitingexample, nucleic acid molecules are allowed to hybridize in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morelow stringency washes in 0.2× SSC/0.1% SDS at room temperature, or byone or more moderate stringency washes in 0.2× SSC/0. 1% SDS at 42° C.,or washed in 0.2× SSC/0.1% SDS at 65° C. for high stringency. In oneembodiment, an isolated nucleic acid molecule that hybridizes understringent conditions to the sequence of SEQ ID NO:1 corresponds to anaturally-occurring nucleic acid molecule. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein).

[0190] As understood by those of ordinary skill, the exact conditionscan be determined empirically and depend on ionic strength, temperatureand the concentration of destabilizing agents such as formamide ordenaturing agents such as SDS. Other factors considered in determiningthe desired hybridization conditions include the length of the nucleicacid sequences, base composition, percent mismatch between thehybridizing sequences and the frequency of occurrence of subsets of thesequences within other non-identical sequences. Thus, equivalentconditions can be determined by varying one or more of these parameterswhile maintaining a similar degree of identity or similarity between thetwo nucleic acid molecules.

[0191] The present invention also provides isolated nucleic acids thatcontain a single or double stranded fragment or portion that hybridizesunder stringent conditions to the nucleotide sequence of SEQ ID NO:2 orthe complement of SEQ ID NO:2. In one embodiment, the nucleic acidconsists of a portion of the nucleotide sequence of SEQ ID NO:2 and thecomplement of SEQ ID NO:2. The nucleic acid fragments of the inventionare at least about 15, preferably at least about 18, 20, 23 or 25nucleotides, and can be 30, 40, 50, 100, 200, 500 or more nucleotides inlength. Longer fragments, for example, 30 or more nucleotides in length,which encode antigenic proteins or polypeptides described herein areuseful.

[0192] As discussed, the invention provides polynucleotides thatcomprise a fragment of the full-length carboxypeptidase polynucleotides.The fragment can be single or double-stranded and can comprise DNA orRNA. The fragment can be derived from either the coding or thenon-coding sequence.

[0193] In another embodiment an isolated carboxypeptidase nucleic acidencodes the entire coding region. In another embodiment the isolatedcarboxypeptidase nucleic acid encodes a sequence corresponding to themature protein that may be from about amino acid 6 to the last aminoacid. Other fragments include nucleotide sequences encoding the aminoacid fragments described herein.

[0194] Thus, carboxypeptidase nucleic acid fragments further includesequences corresponding to the regions/domains described herein,subregions also described, and specific functional sites.Carboxypeptidase nucleic acid fragments also include combinations of theregions/domains, segments, and other functional sites described above. Aperson of ordinary skill in the art would be aware of the manypermutations that are possible.

[0195] Where the location of the domains or sites have been predicted bycomputer analysis, one of ordinary sill would appreciate that the aminoacid residues constituting these domains can vary depending on thecriteria used to define the domains.

[0196] However, it is understood that a carboxypeptidase fragmentincludes any nucleic acid sequence that does not include the entiregene.

[0197] The invention also provides carboxypeptidase nucleic acidfragments that encode epitope bearing regions of the carboxypeptidaseproteins described herein.

[0198] Fragments from about 1-660 can comprise about 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, or morenucleotides.

[0199] Nucleic acid fragments, according to the present invention, arenot to be construed as encompassing those fragments that may have beendisclosed prior to the invention.

[0200] Polynucleotide Uses

[0201] The nucleotide sequences of the present invention can be used asa “query sequence” to perform a search against public databases, forexample, to identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (1997)Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0202] The nucleic acid fragments of the invention provide probes orprimers in assays such as those described below. “Probes” areoligonucleotides that hybridize in a base-specific manner to acomplementary strand of nucleic acid. Such probes include polypeptidenucleic acids, as described in Nielsen et al. (1991) Science254:1497-1500. Typically, a probe comprises a region of nucleotidesequence that hybridizes under highly stringent conditions to at leastabout 15, typically about 20-25, and more typically about 40, 50 or 75consecutive nucleotides of the nucleic acid sequence shown in SEQ IDNO:2 and the complements thereof. More typically, the probe furthercomprises a label, e.g., radioisotope, fluorescent compound, enzyme, orenzyme co-factor.

[0203] As used herein, the term “primer” refers to a single-strandedoligonucleotide which acts as a point of initiation of template-directedDNA synthesis using well-known methods (e.g., PCR, LCR) including, butnot limited to those described herein. The appropriate length of theprimer depends on the particular use, but typically ranges from about 15to 30 nucleotides. The term “primer site” refers to the area of thetarget DNA to which a primer hybridizes. The term “primer pair” refersto a set of primers including a 5′ (upstream) primer that hybridizeswith the 5′ end of the nucleic acid sequence to be amplified and a 3′(downstream) primer that hybridizes with the complement of the sequenceto be amplified.

[0204] The carboxypeptidase polynucleotides are thus useful for probes,primers, and in biological assays.

[0205] Where the polynucleotides are used to assess carboxypeptidaseproperties or functions, such as in the assays described herein, all orless than all of the entire cDNA can be useful. Assays specificallydirected to carboxypeptidase functions, such as assessing agonist orantagonist activity, encompass the use of known fragments. Further,diagnostic methods for assessing carboxypeptidase function can also bepracticed with any fragment, including those fragments that may havebeen known prior to the invention. Similarly, in methods involvingtreatment of carboxypeptidase dysfunction, all fragments are encompassedincluding those, which may have been known in the art.

[0206] The carboxypeptidase polynucleotides are useful as ahybridization probe for cDNA and genomic DNA to isolate a full-lengthcDNA and genomic clones encoding the polypeptides described in SEQ IDNO:1 and to isolate cDNA and genomic clones that correspond to variantsproducing the same polypeptides shown in SEQ ID NO:1 or the othervariants described herein. Variants can be isolated from the same tissueand organism from which the polypeptides shown in SEQ ID NO:1 wereisolated, different tissues from the same organism, or from differentorganisms. This method is useful for isolating genes and cDNA that aredevelopmentally-controlled and therefore may be expressed in the sametissue or different tissues at different points in the development of anorganism.

[0207] The probe can correspond to any sequence along the entire lengthof the gene encoding the carboxypeptidase. Accordingly, it could bederived from 5′ noncoding regions, the coding region, and 3′ noncodingregions.

[0208] The nucleic acid probe can be, for example, the full-length cDNAof SEQ ID NO:2, or a fragment thereof, such as an oligonucleotide of atleast 12, 15, 30, 50, 100, 250 or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions to mRNAor DNA.

[0209] Fragments of the polynucleotides described herein are also usefulto synthesize larger fragments or full-length polynucleotides describedherein. For example, a fragment can be hybridized to any portion of anMRNA and a larger or full-length cDNA can be produced.

[0210] The fragments are also useful to synthesize antisense moleculesof desired length and sequence.

[0211] Antisense nucleic acids of the invention can be designed usingthe nucleotide sequences of SEQ ID NO:2, and constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest).

[0212] Additionally, the nucleic acid molecules of the invention can bemodified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4:5). As used herein, theterms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics,e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrupet al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl Acad. Sci.USA 93:14670. PNAs can be further modified, e.g., to enhance theirstability, specificity or cellular uptake, by attaching lipophilic orother helper groups to PNA, by the formation of PNA-DNA chimeras, or bythe use of liposomes or other techniques of drug delivery known in theart. The synthesis 6f PNA-DNA chimeras can be performed as described inHyrup (1996), supra, Finn et al. (1996) Nucleic Acids Res.24(17):3357-63, Mag et al. (1989) Nucleic Acids Res. 17:5973, andPeterser et al. (1975) Bioorganic Med. Chem. Lett. 5:1119.

[0213] The nucleic acid molecules and fragments of the invention canalso include other appended groups such as peptides (e.g., for targetinghost cell carboxypeptidases in vivo), or agents facilitating transportacross the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl.Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad.Sci. USA 84:648-652; PCT Publication No. WO 88/0918) or the blood brainbarrier (see, e.g., PCT Publication No. WO 89/10134). In addition,oligonucleotides can be modified with hybridization-triggered cleavageagents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) orintercalating agents (see, e.g., Zon (1988) Pharm Res. 5:539-549).

[0214] The carboxypeptidase polynucleotides are also useful as primersfor PCR to amplify any given region of a carboxypeptidasepolynucleotide.

[0215] The carboxypeptidase polynucleotides are also useful forconstructing recombinant vectors. Such vectors include expressionvectors that express a portion of, or all of, the carboxypeptidasepolypeptides. Vectors also include insertion vectors, used to integrateinto another polynucleotide sequence, such as into the cellular genome,to alter in situ expression of carboxypeptidase genes and gene products.For example, an endogenous carboxypeptidase coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

[0216] The carboxypeptidase polynucleotides are also useful forexpressing antigenic portions of the carboxypeptidase proteins.

[0217] The carboxypeptidase polynucleotides are also useful as probesfor determining the chromosomal positions of the carboxypeptidasepolynucleotides by means of in situ hybridization methods, such as FISH.(For a review of this technique, see Verma et al. (1988) HumanChromosomes: A Manual of Basic Techniques (Pergamon Press, New York),and PCR mapping of somatic cell hybrids. The mapping of the sequences tochromosomes is an important first step in correlating these sequenceswith genes associated with disease.

[0218] Reagents for chromosome mapping can be used individually to marka single chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

[0219] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. (Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man, available on-line throughJohns Hopkins University Welch Medical Library). The relationshipbetween a gene and a disease mapped to the same chromosomal region, canthen be identified through linkage analysis (co-inheritance ofphysically adjacent genes), described in, for example, Egeland et al.((1987) Nature 325:783-787).

[0220] Moreover, differences in the DNA sequences between individualsaffected with or free of a disease associated with a specified gene, canbe determined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations, that are visible from chromosome spreads, or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

[0221] The carboxypeptidase polynucleotide probes are also useful todetermine patterns of the presence of the gene encoding thecarboxypeptidases and their variants with respect to tissuedistribution, for example, whether gene duplication has occurred andwhether the duplication occurs in all or only a subset of tissues. Thegenes can be naturally occurring or can have been introduced into acell, tissue, or organism exogenously.

[0222] The carboxypeptidase polynucleotides are also useful fordesigning ribozymes corresponding to all, or a part, of the MRNAproduced from genes encoding the polynucleotides described herein.

[0223] The carboxypeptidase polynucleotides are also useful forconstructing host cells expressing a part, or all, of thecarboxypeptidase polynucleotides and polypeptides.

[0224] The carboxypeptidase polynucleotides are also useful forconstructing transgenic animals expressing all, or a part, of thecarboxypeptidase polynucleotides and polypeptides.

[0225] The carboxypeptidase polynucleotides are also useful for makingvectors that express part, or all, of the carboxypeptidase polypeptides.

[0226] The carboxypeptidase polynucleotides are also useful ashybridization probes for determining the level of carboxypeptidasenucleic acid expression. Accordingly, the probes can be used to detectthe presence of, or to determine levels of, carboxypeptidase nucleicacid in cells, tissues, and in organisms. The nucleic acid whose levelis determined can be DNA or RNA. Accordingly, probes corresponding tothe polypeptides described herein can be used to assess gene copy numberin a given cell, tissue, or organism. This is particularly relevant incases in which there has been an amplification of the carboxypeptidasegenes.

[0227] Alternatively, the probe can be used in an in situ hybridizationcontext to assess the position of extra copies of the carboxypeptidasegenes, as on extrachromosomal elements or as integrated into chromosomesin which the carboxypeptidase gene is not normally found, for example asa homogeneously staining region.

[0228] These uses are relevant for diagnosis of disorders involving anincrease or decrease in carboxypeptidase expression relative to normal,such as a proliferative disorder, a differentiative or developmentaldisorder, or a hematopoietic disorder.

[0229] Disorders in which the carboxypeptidase expression is relevantinclude, but are not limited to, breast, colon and lung carcinoma, andto disorders involving the tissues in which the gene is expressed,especially as disclosed herein.

[0230] Thus, the present invention provides a method for identifying adisease or disorder associated with aberrant expression or activity ofcarboxypeptidase nucleic acid, in which a test sample is obtained from asubject and nucleic acid (e.g., mRNA, genomic DNA) is detected, whereinthe presence of the nucleic acid is diagnostic for a subject having orat risk of developing a disease or disorder associated with aberrantexpression or activity of the nucleic acid.

[0231] One aspect of the invention relates to diagnostic assays fordetermining nucleic acid expression as well as activity in the contextof a biological sample (e.g., blood, serum, cells, tissue) to determinewhether an individual has a disease or disorder, or is at risk ofdeveloping a disease or disorder, associated with aberrant nucleic acidexpression or activity. Such assays can be used for prognostic orpredictive purpose to thereby prophylactically treat an individual priorto the onset of a disorder characterized by or associated withexpression or activity of the nucleic acid molecules.

[0232] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

[0233] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express the carboxypeptidase, such asby measuring the level of a carboxypeptidase-coding nucleic acid in asample of cells from a subject e.g., mRNA or genomic DNA, or determiningif the carboxypeptidase gene has been mutated.

[0234] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate carboxypeptidase nucleic acidexpression (e.g., antisense, polypeptides, peptidomimetics, smallmolecules or other drugs). A cell is contacted with a candidate compoundand the expression of mRNA determined. The level of expression of themRNA in the presence of the candidate compound is compared to the levelof expression of the MRNA in the absence of the candidate compound. Thecandidate compound can then be idenfified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Themodulator can bind to the nucleic acid or indirectly modulateexpression, such as by interacting with other cellular components thataffect nucleic acid expression Modulatory methods can be performed invitro (e.g., by culturing the cell with the agent) or, alternatively, invivo (e.g., by administering the gent to a subject) in patients or intransgenic animals.

[0235] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the carboxypeptidase gene. The method typically includesassaying the ability of the compound to modulate the expression of thecarboxypeptidase nucleic acid and thus identifying a compound that canbe used to treat a disorder characterized by undesired carboxypeptidasenucleic acid expression.

[0236] The assays can be performed in cell-based and cell-free systems.Cell-based assays include cells naturally expressing thecarboxypeptidase nucleic acid or recombinant cells geneticallyengineered to express specific nucleic acid sequences.

[0237] Alternatively, candidate compounds can be assayed in vivo inpatients or in transgenic animals.

[0238] The assay for carboxypeptidase nucleic acid expression caninvolve direct assay of nucleic acid levels, such as mRNA levels, or oncollateral compounds involved in the pathway. Further, the expression ofgenes that are up- or down-regulated in response to the carboxypeptidasepathway can also be assayed. In this embodiment the regulatory regionsof these genes can be operably linked to a reporter gene such asluciferase.

[0239] Thus, modulators of carboxypeptidase gene expression can beidentified in a method wherein a cell is contacted with a candidatecompound and the expression of mRNA determined. The level of expressionof carboxypeptidase mRNA in the presence of the candidate compound iscompared to the level of expression of carboxypeptidase MRNA in theabsence of the candidate compound. The candidate compound can then beidentified as a modulator of nucleic acid expression based on thiscomparison and be used, for example to treat a disorder characterized byaberrant nucleic acid expression. When expression of mRNA isstatistically significantly greater in the presence of the candidatecompound than in its absence, the candidate compound is identified as astimulator of nucleic acid expression. When nucleic acid expression isstatistically significantly less in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of nucleic acid expression.

[0240] Accordingly, the invention provides methods of treatment, withthe nucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate carboxypeptidase nucleic acidexpression. Modulation includes both up-regulation (i.e. activation oragonization) or down-regulation (suppression or antagonization) oreffects on nucleic acid activity (e.g. when nucleic acid is mutated orimproperly modified). Treatment is of disorders characterized byaberrant expression or activity of the nucleic acid.

[0241] Disorders in which the carboxypeptidase expression is relevantinclude, but are not limited to, breast, colon and lung carcinoma, andto disorders involving the tissues in which the gene is expressed,especially as disclosed herein.

[0242] Alternatively, a modulator for carboxypeptidase nucleic acidexpression can be a small molecule or drug identified using thescreening assays described herein as long as the drug or small moleculeinhibits the carboxypeptidase nucleic acid expression.

[0243] The carboxypeptidase polynucleotides are also useful formonitoring the effectiveness of modulating compounds on the expressionor activity of the carboxypeptidase gene in clinical trials or in atreatment regimen. Thus, the gene expression pattern can serve as abarometer for the continuing effectiveness of treatment with thecompound, particularly with compounds to which a patient can developresistance. The gene expression pattern can also serve as a markerindicative of a physiological response of the affected cells to thecompound. Accordingly, such monitoring would allow either increasedadministration of the compound or the administration of alternativecompounds to which the patient has not become resistant. Similarly, ifthe level of nucleic acid expression falls below a desirable level,administration of the compound could be commensurately decreased.

[0244] Monitoring can be, for example, as follows: (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the level of expression of a specified mRNA orgenomic DNA of the invention in the pre-administration sample; (iii)obtaining one or more post-administration samples from the subject; (iv)detecting the level of expression or activity of the mRNA or genomic DNAin the post-administration samples; (v) comparing the level ofexpression or activity of the mRNA or genomic DNA in thepre-administration sample with the MRNA or genomic DNA in thepost-administration sample or samples; and (vi) increasing or decreasingthe administration of the agent to the subject accordingly.

[0245] The carboxypeptidase polynucleotides are also useful indiagnostic assays for qualitative changes in carboxypeptidase nucleicacid, and particularly in qualitative changes that lead to pathology.The polynucleotides can be used to detect mutations in carboxypeptidasegenes and gene expression products such as mRNA. The polynucleotides canbe used as hybridization probes to detect naturally-occurring geneticmutations in the carboxypeptidase gene and thereby to determine whethera subject with the mutation is at risk for a disorder caused by themutation. Mutations include deletion, addition, or substitution of oneor more nucleotides in the gene, chromosomal rearrangement, such asinversion or transposition, modification of genomic DNA, such asaberrant methylation patterns or changes in gene copy number, such asamplification. Detection of a mutated form of the carboxypeptidase geneassociated with a dysfunction provides a diagnostic tool for an activedisease or susceptibility to disease when the disease results fromoverexpression, underexpression, or altered expression of acarboxypeptidase.

[0246] Mutations in the carboxypeptidase gene can be detected at thenucleic acid level by a variety of techniques. Genomic DNA can beanalyzed directly or can be amplified by using PCR prior to analysis.RNA or cDNA can be used in the same way.

[0247] In certain embodiments, detection of the mutation involves theuse of a probe/primer in a polymerase chain reaction (PCR) (see, e.g.U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR,or, alternatively, in a ligation chain reaction (LCR) (see, e.g.,Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al.(1994) PNAS 91:360-364), the latter of which can be particularly usefulfor detecting point mutations in the gene (see Abravaya et al. (1995)Nucleic Acids Res. 23:675-682). This method can include the steps ofcollecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

[0248] It is anticipated that PCR and/or LCR may be desirable to use asa preliminary amplification step in conjunction with any of thetechniques used for detecting mutations described herein.

[0249] Alternative amplification methods include: self sustainedsequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well-known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

[0250] Alternatively, mutations in a carboxypeptidase gene can bedirectly identified, for example, by alterations in restriction enzymedigestion patterns determined by gel electrophoresis.

[0251] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

[0252] Perfectly matched sequences can be distinguished from mismatchedsequences by nuclease cleavage digestion assays or by differences inmelting temperature.

[0253] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and SI protection or thechemical cleavage method.

[0254] Furthermore, sequence differences between a mutantcarboxypeptidase gene and a wild-type gene can be determined by directDNA sequencing. A variety of automated sequencing procedures can beutilized when performing the diagnostic assays ((1995) Biotechniques19:448), including sequencing by mass spectrometry (see, e.g., PCTInternational Publication No. WO 94/16101; Cohen et al. (1996) Adv.Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem.Biotechnol. 38:147-159).

[0255] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al. (1985) Science230:1242); Cotton et al. (1988) PNAS 85:4397; Saleeba et al. (1992)Meth. Enzymol. 21 7:286-295), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al. (1989) PNAS 86:2766;Cotton et al. (1993) Mutat. Res. 285:125-144; and Hayashi et al. (1992)Genet. Anal. Tech. Appl. 9:73-79), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.(1985) Nature 313:495). The sensitivity of the assay may be enhanced byusing RNA (rather than DNA), in which the secondary structure is moresensitive to a change in sequence. In one embodiment, the subject methodutilizes heteroduplex analysis to separate double stranded heteroduplexmolecules on the basis of changes in electrophoretic mobility (Keen etal. (1991) Trends Genet. 7:5). Examples of other techniques fordetecting point mutations include, selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0256] In other embodiments, genetic mutations can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotideprobes (Cronin et al. (1996) Human Mutation 7:244-255; Kozal et al.(1996) Nature Medicine 2:753-759). For example, genetic mutations can beidentified in two dimensional arrays containing light-generated DNAprobes as described in Cronin et al. supra. Briefly, a firsthybridization array of probes can be used to scan through long stretchesof DNA in a sample and control to identify base changes between thesequences by making linear arrays of sequential overlapping probes. Thisstep allows the identification of point mutations. This step is followedby a second hybridization array that allows the characterization ofspecific mutations by using smaller, specialized probe arrayscomplementary to all variants or mutations detected. Each mutation arrayis composed of parallel probe sets, one complementary to the wild-typegene and the other complementary to the mutant gene.

[0257] The carboxypeptidase polynucleotides are also useful for testingan individual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, thepolynucleotides can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). In the present case, forexample, a mutation in the carboxypeptidase gene that results in alteredaffinity for substrate could result in an excessive or decreased drugeffect with standard concentrations of substrate. Accordingly, thecarboxypeptidase polynucleotides described herein can be used to assessthe mutation content of the gene in an individual in order to select anappropriate compound or dosage regimen for treatment.

[0258] Thus polynucleotides displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0259] The methods can involve obtaining a control biological samplefrom a control subject, contacting the control sample with a compound oragent capable of detecting mRNA, or genomic DNA, such that the presenceof mRNA or genomic DNA is detected in the biological sample, andcomparing the presence of mRNA or genomic DNA in the control sample withthe presence of mRNA or genomic DNA in the test sample.

[0260] The carboxypeptidase polynucleotides are also useful forchromosome identification when the sequence is identified with anindividual chromosome and to a particular location on the chromosome.First, the DNA sequence is matched to the chromosome by in situ or otherchromosome-specific hybridization. Sequences can also be correlated tospecific chromosomes by preparing PCR primers that can be used for PCRscreening of somatic cell hybrids containing individual chromosomes fromthe desired species. Only hybrids containing the chromosome containingthe gene homologous to the primer will yield an amplified fragment.Sublocalization can be achieved using chromosomal fragments. Otherstrategies include prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to chromosome-specific libraries. Furthermapping strategies include fluorescence in situ hybridization, whichallows hybridization with probes shorter than those traditionally used.Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on the chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

[0261] The carboxypeptidase polynucleotides can also be used to identifyindividuals from small biological samples. This can be done for exampleusing restriction fragment-length polymorphism (RFLP) to identify anindividual. Thus, the polynucleotides described herein are useful as DNAmarkers for RFLP (See U.S. Pat. No. 5,272,057).

[0262] Furthermore, the carboxypeptidase sequence can be used to providean alternative technique, which determines the actual DNA sequence ofselected fragments in the genome of an individual. Thus, thecarboxypeptidase sequences described herein can be used to prepare twoPCR primers from the 5′ and 3′ ends of the sequences. These primers canthen be used to amplify DNA from an individual for subsequentsequencing.

[0263] Panels of corresponding DNA sequences from individuals preparedin this manner can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences. It is estimatedthat allelic variation in humans occurs with a frequency of about onceper each 500 bases. Allelic variation occurs to some degree in thecoding regions of these sequences, and to a greater degree in thenoncoding regions. The carboxypeptidase sequences can be used to obtainsuch identification sequences from individuals and from tissue. Thesequences represent unique fragments of the human genome. Each of thesequences described herein can, to some degree, be used as a standardagainst which DNA from an individual can be compared for identificationpurposes.

[0264] If a panel of reagents from the sequences is used to generate aunique identification database for an individual, those same reagentscan later be used to identify tissue from that individual. Using theunique identification database, positive identification of theindividual, living or dead, can be made from extremely small tissuesamples.

[0265] The carboxypeptidase polynucleotides can also be used in forensicidentification procedures. PCR technology can be used to amplify DNAsequences taken from very small biological samples, such as a singlehair follicle, body fluids (e.g. blood, saliva, or semen). The amplifiedsequence can then be compared to a standard allowing identification ofthe origin of the sample.

[0266] The carboxypeptidase polynucleotides can thus be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As described above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to the noncoding region are particularly useful since greaterpolymorphism occurs in the noncoding regions, making it easier todifferentiate individuals using this technique.

[0267] The carboxypeptidase polynucleotides can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue. This is useful in cases in which a forensicpathologist is presented with a tissue of unknown origin. Panels ofcarboxypeptidase probes can be used to identify tissue by species and/orby organ type.

[0268] In a similar fashion, these primers and probes can be used toscreen tissue culture for contamination (i.e. screen for the presence ofa mixture of different types of cells in a culture).

[0269] Alternatively, the carboxypeptidase polynucleotides can be useddirectly to block transcription or translation of carboxypeptidase genesequences by means of antisense or ribozyme constructs. Thus, in adisorder characterized by abnormally high or undesirablecarboxypeptidase gene expression, nucleic acids can be directly used fortreatment.

[0270] The carboxypeptidase polynucleotides are thus useful as antisenseconstructs to control carboxypeptidase gene expression in cells,tissues, and organisms. A DNA antisense polynucleotide is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of carboxypeptidaseprotein. An antisense RNA or DNA polynucleotide would hybridize to themRNA and thus block translation of MRNA into carboxypeptidase protein.

[0271] Examples of antisense molecules useful to inhibit nucleic acidexpression include antisense molecules complementary to a fragment ofthe 5′ untranslated region of SEQ ID NO:2 which also includes the startcodon and antisense molecules which are complementary to a fragment ofthe 3′ untranslated region of SEQ ID NO:2. Alternatively, a class ofantisense molecules can be used to inactivate mRNA in order to decreaseexpression of carboxypeptidase nucleic acid. Accordingly, thesemolecules can treat a disorder characterized by abnormal or undesiredcarboxypeptidase nucleic acid expression. This technique involvescleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the MRNA that attenuate theability of the mRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the carboxypeptidase protein.

[0272] The carboxypeptidase polynucleotides also provide vectors forgene therapy in patients containing cells that are aberrant incarboxypeptidase gene expression. Thus, recombinant cells, which includethe patient's cells that have been engineered ex vivo and returned tothe patient, are introduced into an individual where the cells producethe desired carboxypeptidase protein to treat the individual.

[0273] The invention also encompasses kits for detecting the presence ofa carboxypeptidase nucleic acid in a biological sample. For example, thekit can comprise reagents such as a labeled or labelable nucleic acid oragent capable of detecting carboxypeptidase nucleic acid in a biologicalsample; means for determining the amount of carboxypeptidase nucleicacid in the sample; and means for comparing the amount ofcarboxypeptidase nucleic acid in the sample with a standard. Thecompound or agent can be packaged in a suitable container. The kit canfurther comprise instructions for using the kit to detectcarboxypeptidase mRNA or DNA.

[0274] Computer Readable Means

[0275] The nucleotide or amino acid sequences of the invention are alsoprovided in a variety of mediums to facilitate use thereof. As usedherein, “provided” refers to a manufacture, other than an isolatednucleic acid or amino acid molecule, which contains a nucleotide oramino acid sequence of the present invention. Such a manufactureprovides the nucleotide or amino acid sequences, or a subset thereof(e.g., a subset of open reading frames (ORFs)) in a form which allows askilled artisan to examine the manufacture using means not directlyapplicable to examining the nucleotide or amino acid sequences, or asubset thereof, as they exists in nature or in purified form.

[0276] In one application of this embodiment, a nucleotide or amino acidsequence of the present invention can be recorded on computer readablemedia. As used herein, “computer readable media” refers to any mediumthat can be read and accessed directly by a computer. Such mediainclude, but are not limited to: magnetic storage media, such as floppydiscs, hard disc storage medium, and magnetic tape; optical storagemedia such as CD-ROM; electrical storage media such as RAM and ROM; andhybrids of these categories such as magnetic/optical storage media. Theskilled artisan will readily appreciate how any of the presently knowncomputer readable mediums can be used to create a manufacture comprisingcomputer readable medium having recorded thereon a nucleotide or aminoacid sequence of the present invention.

[0277] As used herein, “recorded” refers to a process for storinginformation on computer readable medium. The skilled artisan can readilyadopt any of the presently known methods for recording information oncomputer readable medium to generate manufactures comprising thenucleotide or amino acid sequence information of the present invention.

[0278] A variety of data storage structures are available to a skilledartisan for creating a computer readable medium having recorded thereona nucleotide or amino acid sequence of the present invention. The choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium. The sequence information can be represented in a word processingtext file, formatted in commercially-available software such asWordPerfect and Microsoft Word, or represented in the form of an ASCIIfile, stored in a database application, such as DB2, Sybase, Oracle, orthe like. The skilled artisan can readily adapt any number ofdataprocessor structuring formats (e.g., text file or database) in orderto obtain computer readable medium having recorded thereon thenucleotide sequence information of the present invention.

[0279] By providing the nucleotide or amino acid sequences of theinvention in computer readable form, the skilled artisan can routinelyaccess the sequence information for a variety of purposes. For example,one skilled in the art can use the nucleotide or amino acid sequences ofthe invention in computer readable form to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. Search means are used to identify fragments orregions of the sequences of the invention which match a particulartarget sequence or target motif.

[0280] As used herein, a “target sequence” can be any DNA or amino acidsequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. The most preferred sequence length of atarget sequence is from about 10 to 100 amino acids or from about 30 to300 nucleotide residues. However, it is well recognized thatcommercially important fragments, such as sequence fragments involved ingene expression and protein processing, may be of shorter length.

[0281] As used herein, “a target structural motif,” or “target motif,”refers to any rationally selected sequence or combination of sequencesin which the sequence(s) are chosen based on a three-dimensionalconfiguration which is formed upon the folding of the target motif.There are a variety of target motifs known in the art. Protein targetmotifs include, but are not limited to, enzyme active sites and signalsequences. Nucleic acid target motifs include, but are not limited to,promoter sequences, hairpin structures and inducible expression elements(protein binding sequences).

[0282] Computer software is publicly available which allows a skilledartisan to access sequence information provided in a computer readablemedium for analysis and comparison to other sequences. A variety ofknown algorithms are disclosed publicly and a variety of commerciallyavailable software for conducting search means are and can be used inthe computer-based systems of the present invention. Examples of suchsoftware includes, but is not limited to, MacPattern (EMBL), BLASTN andBLASTX (NCBIA).

[0283] For example, software which implements the BLAST (Altschul et al.(1990) J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al. (1993) Comp.Chem. 17:203-207) search algorithms on a Sybase system can be used toidentify open reading frames (ORFs) of the sequences of the inventionwhich contain homology to ORFs or proteins from other libraries. SuchORFs are protein encoding fragments and are useful in producingcommercially important proteins such as enzymes used in variousreactions and in the production of commercially useful metabolites.Vectors/Host Cells The invention also provides vectors containing thecarboxypeptidase polynucleotides. The term “vector” refers to a vehicle,preferably a nucleic acid molecule that can transport thecarboxypeptidase polynucleotides. When the vector is a nucleic acidmolecule, the carboxypeptidase polynucleotides are covalently linked tothe vector nucleic acid. With this aspect of the invention, the vectorincludes a plasmid, single or double stranded phage, a single or doublestranded RNA or DNA viral vector, or artificial chromosome, such as aBAC, PAC, YAC, OR MAC.

[0284] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the carboxypeptidase polynucleotides. Alternatively, thevector may integrate into the host cell genome and produce additionalcopies of the carboxypeptidase polynucleotides when the host cellreplicates.

[0285] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of thecarboxypeptidase polynucleotides. The vectors can function inprocaryotic or eukaryotic cells or in both (shuttle vectors).

[0286] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the carboxypeptidase polynucleotidessuch that transcription of the polynucleotides is allowed in a hostcell. The polynucleotides can be introduced into the host cell with aseparate polynucleotide capable of affecting transcription. Thus, thesecond polynucleotide may provide a trans-acting factor interacting withthe cis-regulatory control region to allow transcription of thecarboxypeptidase polynucleotides from the vector. Alternatively, atrans-acting factor may be supplied by the host cell. Finally, atrans-acting factor can be produced from the vector itself.

[0287] It is understood, however, that in some embodiments,transcription and/or translation of the carboxypeptidase polynucleotidescan occur in a cell-free system.

[0288] The regulatory sequence to which the polynucleotides describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

[0289] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0290] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual 2nd. ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

[0291] A variety of expression vectors can be used to express acarboxypeptidase polynucleotide. Such vectors include chromosomal,episomal, and virus-derived vectors, for example vectors derived frombacterial plasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[0292] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0293] The carboxypeptidase polynucleotides can be inserted into thevector nucleic acid by well-known methodology. Generally, the DNAsequence that will ultimately be expressed is joined to an expressionvector by cleaving the DNA sequence and the expression vector with oneor more restriction enzymes and then ligating the fragments together.Procedures for restriction enzyme digestion and ligation are well knownto those of ordinary skill in the art.

[0294] The vector containing the appropriate polynucleotide can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0295] As described herein, it may be desirable to express thepolypeptide as a fusion protein. Accordingly, the invention providesfusion vectors that allow for the production of the carboxypeptidasepolypeptides. Fusion vectors can increase the expression of arecombinant protein, increase the solubility of the recombinant protein,and aid in the purification of the protein by acting for example as aligand for affinity purification. A proteolytic cleavage site may beintroduced at the junction of the fusion moiety so that the desiredpolypeptide can ultimately be separated from the fusion moiety.Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enterokinase. Typical fusion expression vectors includepGEX (Smith et al. (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal. (1988) Gene 69:301-315) and pET 11d (Studier et al. (1990) GeneExpression Technology: Methods in Enzymology 185:60-89).

[0296] Recombinant protein expression can be maximized in a hostbacteria by providing a genetic background wherein the host cell has animpaired capacity to proteolytically cleave the recombinant protein.(Gottesman, S. (1990) Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. 119-128). Alternatively, thesequence of the polynucleotide of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al. (1992) Nucleic Acids Res. 20:2111-2118).

[0297] The carboxypeptidase polynucleotides can also be expressed byexpression vectors that are operative in yeast. Examples of vectors forexpression in yeast e.g., S. cerevisiae include pYepSec1 (Baldari et al.(1987) EMBO J. 6:229-234), pMFa (Kurjan et al. (1982) Cell 30:933-943),pJRY88 (Schultz et al. (1987) Gene 54:113-123), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0298] The carboxypeptidase polynucleotides can also be expressed ininsect cells using, for example, baculovirus expression vectors.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow et al.(1989) Virology 170:31-39).

[0299] In certain embodiments of the invention, the polynucleotidesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)EMBO J. 6:187-195).

[0300] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the carboxypeptidasepolynucleotides. The person of ordinary skill in the art would be awareof other vectors suitable for maintenance propagation or expression ofthe polynucleotides described herein. These are found for example inSambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2nd, ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

[0301] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the polynucleotide sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0302] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0303] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

[0304] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the carboxypeptidase polynucleotides can be introducedeither alone or with other polynucleotides that are not related to thecarboxypeptidase polynucleotides such as those providing trans-actingfactors for expression vectors. When more than one vector is introducedinto a cell, the vectors can be introduced independently, co-introducedor joined to the carboxypeptidase polynucleotide vector.

[0305] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0306] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the polynucleotides described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0307] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0308] Where secretion of the polypeptide is desired, appropriatesecretion signals are incorporated into the vector. The signal sequencecan be endogenous to the carboxypeptidase polypeptides or heterologousto these polypeptides. Where the polypeptide is not secreted into themedium, the protein can be isolated from the host cell by standarddisruption procedures, including freeze thaw, sonication, mechanicaldisruption, use of lysing agents and the like. The polypeptide can thenbe recovered and purified by well-known purification methods includingammonium sulfate precipitation, acid extraction, anion or cationicexchange chromatography, phosphocellulose chromatography,hydrophobic-interaction chromatography, affinity chromatography,hydroxylapatite chromatography, lectin chromatography, or highperformance liquid chromatography.

[0309] It is also understood that depending upon the host cell inrecombinant production of the polypeptides described herein, thepolypeptides can have various glycosylation patterns, depending upon thecell, or maybe non-glycosylated as when produced in bacteria. Inaddition, the polypeptides may include an initial modified methionine insome cases as a result of a host-mediated process.

[0310] Uses of Vectors and Host Cells

[0311] It is understood that “host cells” and “recombinant host cells”refer not only to the particular subject cell but also to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

[0312] The host cells expressing the polypeptides described herein, andparticularly recombinant host cells, have a variety of uses. First, thecells are useful for producing carboxypeptidase proteins or polypeptidesthat can be further purified to produce desired amounts ofcarboxypeptidase protein or fragments. Thus, host cells containingexpression vectors are useful for polypeptide production.

[0313] Host cells are also useful for conducting cell-based assaysinvolving the carboxypeptidase or carboxypeptidase fragments. Thus, arecombinant host cell expressing a native carboxypeptidase is useful toassay for compounds that stimulate or inhibit carboxypeptidase function.

[0314] Host cells are also useful for identifying carboxypeptidasemutants in which these functions are affected. If the mutants naturallyoccur and give rise to a pathology, host cells containing the mutationsare useful to assay compounds that have a desired effect on the mutantcarboxypeptidase (for example, stimulating or inhibiting function) whichmay not be indicated by their effect on the native carboxypeptidase.

[0315] Recombinant host cells are also useful for expressing thechimeric polypeptides described herein to assess compounds that activateor suppress activation by means of a heterologous domain, segment, site,and the like, as disclosed herein.

[0316] Further, mutant carboxypeptidases can be designed in which one ormore of the various functions is engineered to be increased or decreased(e.g., peptide substrate binding) and used to augment or replacecarboxypeptidase proteins in an individual. Thus, host cells can providea therapeutic benefit by replacing an aberrant carboxypeptidase orproviding an aberrant carboxypeptidase that provides a therapeuticresult. In one embodiment, the cells provide carboxypeptidases that areabnormally active.

[0317] In another embodiment, the cells provide carboxypeptidases thatare abnormally inactive. These carboxypeptidases can compete withendogenous carboxypeptidases in the individual.

[0318] In another embodiment, cells expressing carboxypeptidases thatcannot be activated, are introduced into an individual in order tocompete with endogenous carboxypeptidases for substrate. For example, inthe case in which excessive substrate is part of a treatment modality,it may be necessary to inactivate this molecule at a specific point intreatment. Providing cells that compete for the molecule, but whichcannot be affected by carboxypeptidase activation would be beneficial.

[0319] Homologously recombinant host cells can also be produced thatallow the in situ alteration of endogenous carboxypeptidasepolynucleotide sequences in a host cell genome. This technology is morefully described in WO 93/09222, WO 91/12650 and U.S. Pat. No. 5,641,670.Briefly, specific polynucleotide sequences corresponding to thecarboxypeptidase polynucleotides or sequences proximal or distal to acarboxypeptidase gene are allowed to integrate into a host cell genomeby homologous recombination where expression of the gene can beaffected. In one embodiment, regulatory sequences are introduced thateither increase or decrease expression of an endogenous sequence.Accordingly, a carboxypeptidase protein can be produced in a cell notnormally producing it, or increased expression of carboxypeptidaseprotein can result in a cell normally producing the protein at aspecific level. Alternatively, the entire gene can be deleted. Stillfurther, specific mutations can be introduced into any desired region ofthe gene to produce mutant carboxypeptidase proteins. Such mutationscould be introduced, for example, into the specific regions disclosedherein.

[0320] In one embodiment, the host cell can be a fertilized oocyte orembryonic stem cell that can be used to produce a transgenic animalcontaining the altered carboxypeptidase gene. Alternatively, the hostcell can be a stem cell or other early tissue precursor that gives riseto a specific subset of cells and can be used to produce transgenictissues in an animal. See also Thomas et al., Cell 51:503 (1987) for adescription of homologous recombination vectors. The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced gene has homologously recombined withthe endogenous carboxypeptidase gene is selected (see e.g., Li, E. etal. (1992) Cell 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: APractical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp.113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term.Progeny harboring the homologously recombined DNA in their germ cellscan be used to breed animals in which all cells of the animal containthe homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described further in Bradley, A.(1991) Current Opinion in Biotechnology 2:823-829 and in PCTInternational Publication Nos. WO 90/11354; WO 91/01140; and WO93/04169.

[0321] The genetically engineered host cells can be used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a carboxypeptidaseprotein and identifying and evaluating modulators of carboxypeptidaseprotein activity.

[0322] Other examples of transgenic animals include non-human primates,sheep, dogs, cows, goats, chickens, and amphibians.

[0323] In one embodiment, a host cell is a fertilized oocyte or anembryonic stem cell into which carboxypeptidase polynucleotide sequenceshave been introduced.

[0324] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the carboxypeptidasenucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0325] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the carboxypeptidase protein toparticular cells.

[0326] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0327] In another embodiment, transgenic non-human animals can beproduced which contain selected systems, which allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. (1992) PNAS89:6232-6236. Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. (1991) Science251:1351-1355. If a cre/loxP recombinase system is used to regulateexpression of the transgene, animals containing transgenes encoding boththe Cre recombinase and a selected protein is required. Such animals canbe provided through the construction of “double” transgenic animals,e.g., by mating two transgenic animals, one containing a transgeneencoding a selected protein and the other containing a transgeneencoding a recombinase.

[0328] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut et al.(1997) Nature 385:810-813 and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(O) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to a pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0329] Transgenic animals containing recombinant cells that express thepolypeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could affect, for example,substrate binding and hydrolysis or carboxypeptidase activation, may notbe evident from in vitro cell-free or cell-based assays. Accordingly, itis useful to provide non-human transgenic animals to assay in vivocarboxypeptidase function, including substrate interaction, the effectof specific mutant carboxypeptidases on carboxypeptidase function andsubstrate interaction, and the effect of chimeric carboxypeptidases. Itis also possible to assess the effect of null mutations, that ismutations that substantially or completely eliminate one or morecarboxypeptidase functions.

[0330] Pharmaceutical Compositions

[0331] The carboxypeptidase nucleic acid molecules, protein, modulatorsof the protein, and antibodies (also referred to herein as “activecompounds”) can be incorporated into pharmaceutical compositionssuitable for administration to a subject, e.g., a human. Suchcompositions typically comprise the nucleic acid molecule, protein,modulator, or antibody and a pharmaceutically acceptable carrier.

[0332] The term “administer” is used in its broadest sense and includesany method of introducing the compositions of the present invention intoa subject. This includes producing polypeptides or polynucleotides invivo as by transcription or translation in vivo of polynucleotides thathave been exogenously introduced into a subject. Thus, polypeptides ornucleic acids produced in the subject from the exogenous compositionsare encompassed in the term “administer.”

[0333] As used herein the language “pharmaceutically acceptable carrier”is intended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, such media can be used in thecompositions of the invention. Supplementary active compounds can alsobe incorporated into the compositions. A pharmaceutical composition ofthe invention is formulated to be compatible with its intended route ofadministration. Examples of routes of administration include parenteral,e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampules, disposable syringesor multiple dose vials made of glass or plastic.

[0334] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0335] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., a carboxypeptidase protein or anti-carboxypeptidase antibody) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

[0336] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For oral administration, the agent can be contained in entericforms to survive the stomach or further coated or mixed to be releasedin a particular region of the GI tract by known methods. For the purposeof oral therapeutic administration, the active compound can beincorporated with excipients and used in the form of tablets, troches,or capsules. Oral compositions can also be prepared using a fluidcarrier for use as a mouthwash, wherein the compound in the fluidcarrier is applied orally and swished and expectorated or swallowed.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0337] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser,which contains a suitable propellant, e.g., a gas such as carbondioxide, or a nebulizer.

[0338] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0339] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0340] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0341] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. “Dosage unit form” as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0342] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470) or by stereotactic injection(see e.g., Chen et al. (1994) PNAS 91:3054-3057). The pharmaceuticalpreparation of the gene therapy vector can include the gene therapyvector in an acceptable diluent, or can comprise a slow release matrixin which the gene delivery vehicle is imbedded. Alternatively, where thecomplete gene delivery vector can be produced intact from recombinantcells, e.g. retroviral vectors, the pharmaceutical preparation caninclude one or more cells which produce the gene delivery system.

[0343] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0344] This invention may be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will filly conveythe invention to those skilled in the art. Many modifications and otherembodiments of the invention will come to mind in one skilled in the artto which this invention pertains having the benefit of the teachingspresented in the foregoing description. Although specific terms areemployed, they are used as in the art unless otherwise indicated.

1 4 1 492 PRT Homo sapiens 1 Met Gly Ser Ala Pro Trp Ala Pro Val Leu LeuLeu Ala Leu Gly Leu 1 5 10 15 Arg Gly Leu Gln Ala Gly Ala Arg Arg AlaPro Asp Pro Gly Phe Gln 20 25 30 Glu Arg Phe Phe Gln Gln Arg Leu Asp HisPhe Asn Phe Glu Arg Phe 35 40 45 Gly Asn Lys Thr Phe Pro Gln Arg Phe LeuVal Ser Asp Arg Phe Trp 50 55 60 Val Arg Gly Glu Gly Pro Ile Phe Phe TyrThr Gly Asn Glu Gly Asp 65 70 75 80 Val Trp Ala Phe Ala Asn Asn Ser GlyPhe Val Ala Glu Leu Ala Ala 85 90 95 Glu Arg Gly Ala Leu Leu Val Phe AlaGlu His Arg Tyr Tyr Gly Lys 100 105 110 Ser Leu Pro Phe Gly Ala Gln SerThr Gln Arg Gly His Thr Glu Leu 115 120 125 Leu Thr Val Glu Gln Ala LeuAla Asp Phe Ala Glu Leu Leu Arg Ala 130 135 140 Leu Arg Arg Asp Leu GlyAla Gln Asp Ala Pro Ala Ile Ala Phe Gly 145 150 155 160 Gly Ser Tyr GlyGly Met Leu Ser Ala Tyr Leu Arg Met Lys Tyr Pro 165 170 175 His Leu ValAla Gly Ala Leu Ala Ala Ser Ala Pro Val Leu Ala Val 180 185 190 Ala GlyLeu Gly Asp Ser Asn Gln Phe Phe Arg Asp Val Thr Ala Asp 195 200 205 PheGlu Gly Gln Ser Pro Lys Cys Thr Gln Gly Val Arg Glu Ala Phe 210 215 220Arg Gln Ile Lys Asp Leu Phe Leu Gln Gly Ala Tyr Asp Thr Val Arg 225 230235 240 Trp Glu Phe Gly Thr Cys Gln Pro Leu Ser Asp Glu Lys Asp Leu Thr245 250 255 Gln Leu Phe Met Phe Ala Arg Asn Ala Phe Thr Val Leu Ala MetMet 260 265 270 Asp Tyr Pro Tyr Pro Thr Asp Phe Leu Gly Pro Leu Pro AlaAsn Pro 275 280 285 Val Lys Val Gly Cys Asp Arg Leu Leu Ser Glu Ala GlnArg Ile Thr 290 295 300 Gly Leu Arg Ala Leu Ala Gly Leu Val Tyr Asn AlaSer Gly Ser Glu 305 310 315 320 His Cys Tyr Asp Ile Tyr Arg Leu Tyr HisSer Cys Ala Asp Pro Thr 325 330 335 Gly Cys Gly Thr Gly Pro Asp Ala ArgAla Trp Asp Tyr Gln Ala Cys 340 345 350 Thr Glu Ile Asn Leu Thr Phe AlaSer Asn Asn Val Thr Asp Met Phe 355 360 365 Pro Asp Leu Pro Phe Thr AspGlu Leu Arg Gln Arg Tyr Cys Leu Asp 370 375 380 Thr Trp Gly Val Trp ProArg Pro Asp Trp Leu Leu Thr Ser Phe Trp 385 390 395 400 Gly Gly Asp LeuArg Ala Ala Ser Asn Ile Ile Phe Ser Asn Gly Asn 405 410 415 Leu Asp ProTrp Ala Gly Gly Gly Ile Arg Arg Asn Leu Ser Ala Ser 420 425 430 Val IleAla Val Thr Ile Gln Gly Gly Ala His His Leu Asp Leu Arg 435 440 445 AlaSer His Pro Glu Asp Pro Ala Ser Val Val Glu Ala Arg Lys Leu 450 455 460Glu Ala Thr Ile Ile Gly Glu Trp Val Lys Ala Ala Arg Arg Glu Gln 465 470475 480 Gln Pro Ala Leu Arg Gly Gly Pro Arg Leu Ser Leu 485 490 2 1653DNA Homo sapiens CDS (19)...(1494) 2 cgtccggcgg aaggcgac atg ggc tcc gctccc tgg gcc ccg gtc ctg ctg 51 Met Gly Ser Ala Pro Trp Ala Pro Val LeuLeu 1 5 10 ctg gcg ctc ggg ctg cgc ggc ctc cag gcg ggg gcc cgc agg gccccg 99 Leu Ala Leu Gly Leu Arg Gly Leu Gln Ala Gly Ala Arg Arg Ala Pro15 20 25 gac ccc ggc ttc cag gag cgc ttc ttc cag cag cgt ctg gac cac ttc147 Asp Pro Gly Phe Gln Glu Arg Phe Phe Gln Gln Arg Leu Asp His Phe 3035 40 aac ttc gag cgc ttc ggc aac aag acc ttc cct cag cgc ttc ctg gtg195 Asn Phe Glu Arg Phe Gly Asn Lys Thr Phe Pro Gln Arg Phe Leu Val 4550 55 tcg gac agg ttc tgg gtc cgg ggc gag ggg ccc atc ttc ttc tac act243 Ser Asp Arg Phe Trp Val Arg Gly Glu Gly Pro Ile Phe Phe Tyr Thr 6065 70 75 ggg aac gag ggc gac gtg tgg gcc ttc gcc aac aac tcg ggc ttc gtc291 Gly Asn Glu Gly Asp Val Trp Ala Phe Ala Asn Asn Ser Gly Phe Val 8085 90 gcg gag ctg gcg gcc gag cgg ggg gct cta ctg gtc ttc gcg gag cac339 Ala Glu Leu Ala Ala Glu Arg Gly Ala Leu Leu Val Phe Ala Glu His 95100 105 cgc tac tac ggg aag tcg ctg ccg ttc ggt gcg cag tcc acg cag cgc387 Arg Tyr Tyr Gly Lys Ser Leu Pro Phe Gly Ala Gln Ser Thr Gln Arg 110115 120 ggg cac acg gag ctg ctg acg gtg gag cag gcc ctg gcc gac ttc gca435 Gly His Thr Glu Leu Leu Thr Val Glu Gln Ala Leu Ala Asp Phe Ala 125130 135 gag ctg ctc cgc gcg cta cga cgc gac ctc ggg gcc cag gat gcc ccc483 Glu Leu Leu Arg Ala Leu Arg Arg Asp Leu Gly Ala Gln Asp Ala Pro 140145 150 155 gcc atc gcc ttc ggt gga agt tat ggg ggg atg ctc agt gcc tacctg 531 Ala Ile Ala Phe Gly Gly Ser Tyr Gly Gly Met Leu Ser Ala Tyr Leu160 165 170 agg atg aag tat ccc cac ctg gtg gcg ggg gcg ctg gcg gcc agcgcg 579 Arg Met Lys Tyr Pro His Leu Val Ala Gly Ala Leu Ala Ala Ser Ala175 180 185 ccc gtt cta gct gtg gca ggc ctc ggc gac tcc aac cag ttc ttccgg 627 Pro Val Leu Ala Val Ala Gly Leu Gly Asp Ser Asn Gln Phe Phe Arg190 195 200 gac gtc acg gcg gac ttt gag ggc cag agt ccc aaa tgc acc cagggt 675 Asp Val Thr Ala Asp Phe Glu Gly Gln Ser Pro Lys Cys Thr Gln Gly205 210 215 gtg cgg gaa gcg ttc cga cag atc aag gac ttg ttc cta cag ggagcc 723 Val Arg Glu Ala Phe Arg Gln Ile Lys Asp Leu Phe Leu Gln Gly Ala220 225 230 235 tac gac acg gtc cgc tgg gag ttc ggc acc tgc cag ccg ctgtca gac 771 Tyr Asp Thr Val Arg Trp Glu Phe Gly Thr Cys Gln Pro Leu SerAsp 240 245 250 gag aag gac ctg acc cag ctc ttc atg ttc gcc cgg aat gccttc acc 819 Glu Lys Asp Leu Thr Gln Leu Phe Met Phe Ala Arg Asn Ala PheThr 255 260 265 gtg ctg gcc atg atg gac tac ccc tac ccc act gac ttc ctgggt ccc 867 Val Leu Ala Met Met Asp Tyr Pro Tyr Pro Thr Asp Phe Leu GlyPro 270 275 280 ctc cct gcc aac ccc gtc aag gtg ggc tgt gat cgg ctg ctgagt gag 915 Leu Pro Ala Asn Pro Val Lys Val Gly Cys Asp Arg Leu Leu SerGlu 285 290 295 gcc cag agg atc acg ggg ctg cga gca ctg gca ggg ctg gtctac aac 963 Ala Gln Arg Ile Thr Gly Leu Arg Ala Leu Ala Gly Leu Val TyrAsn 300 305 310 315 gcc tcg ggc tcc gag cac tgc tac gac atc tac cgg ctctac cac agc 1011 Ala Ser Gly Ser Glu His Cys Tyr Asp Ile Tyr Arg Leu TyrHis Ser 320 325 330 tgt gct gac ccc act ggc tgc ggc acc ggc ccc gac gccagg gcc tgg 1059 Cys Ala Asp Pro Thr Gly Cys Gly Thr Gly Pro Asp Ala ArgAla Trp 335 340 345 gac tac cag gcc tgc acc gag atc aac ctg acc ttc gccagc aac aat 1107 Asp Tyr Gln Ala Cys Thr Glu Ile Asn Leu Thr Phe Ala SerAsn Asn 350 355 360 gtg acc gat atg ttc ccc gac ctg ccc ttc act gac gagctc cgc cag 1155 Val Thr Asp Met Phe Pro Asp Leu Pro Phe Thr Asp Glu LeuArg Gln 365 370 375 cgg tac tgc ctg gac acc tgg ggc gtg tgg ccc cgg cccgac tgg ctg 1203 Arg Tyr Cys Leu Asp Thr Trp Gly Val Trp Pro Arg Pro AspTrp Leu 380 385 390 395 ctg acc agc ttc tgg ggg ggt gat ctc aga gcc gccagc aac atc atc 1251 Leu Thr Ser Phe Trp Gly Gly Asp Leu Arg Ala Ala SerAsn Ile Ile 400 405 410 ttc tcc aac ggg aac ctg gac ccc tgg gca ggg ggcggg att cgg agg 1299 Phe Ser Asn Gly Asn Leu Asp Pro Trp Ala Gly Gly GlyIle Arg Arg 415 420 425 aac ctg agt gcc tca gtc atc gcc gtc acc atc cagggg gga gcg cac 1347 Asn Leu Ser Ala Ser Val Ile Ala Val Thr Ile Gln GlyGly Ala His 430 435 440 cac ctc gac ctc aga gcc tcc cac cca gaa gat cctgct tcc gtg gtt 1395 His Leu Asp Leu Arg Ala Ser His Pro Glu Asp Pro AlaSer Val Val 445 450 455 gag gcg cgg aag ctg gag gcc acc atc atc ggc gagtgg gta aag gca 1443 Glu Ala Arg Lys Leu Glu Ala Thr Ile Ile Gly Glu TrpVal Lys Ala 460 465 470 475 gcc agg cgt gag cag cag cca gct ctg cgt gggggg ccc aga ctc agc 1491 Ala Arg Arg Glu Gln Gln Pro Ala Leu Arg Gly GlyPro Arg Leu Ser 480 485 490 ctc tgagcacagg actggagggg tctcaaggctcctcatggag tgggggcttc 1544 Leu actcaagcag ctggcggcag agggaaggggctgaataaac gcctggaggc ctggcaaaaa 1604 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaa 1653 3 10 PRT Artificial Sequence consensussequence for the prolyloligopeptidase family from the Prosite databaseof protein patterns 3 Ile Phe Gly Gly Ser Asn Gly Gly Leu Leu 1 5 10 4251 PRT Artificial Sequence consensus sequence for the alpha/betahydrolase family from the Prosite database of protein patterns 4 Phe ArgVal Ile Ala Leu Asp Leu Arg Gly Phe Gly Glu Ser Ser Arg 1 5 10 15 ProSer Asp Leu Ala Asp Tyr Arg Phe Asp Asp Leu Ala Glu Asp Leu 20 25 30 GluAla Leu Leu Asp Ala Leu Gly Leu Asp Lys Pro Val Ile Leu Val 35 40 45 GlyHis Ser Met Gly Gly Ala Leu Ala Ala Ala Tyr Ala Ala Lys Tyr 50 55 60 ProGlu Glu Arg Val Lys Ala Leu Val Leu Val Ser Thr Pro Ala Pro 65 70 75 80Ala Gly Leu Ser Ser Arg Leu Phe Pro Arg Leu Gly Asn Leu Glu Gly 85 90 95Leu Leu Leu Ala Asn Phe Phe Asn Arg Leu Ser Arg Ser Val Glu Ala 100 105110 Leu Leu Gly Arg Ala Leu Lys Gln Phe Phe Leu Leu Gly Arg Pro Phe 115120 125 Val Ser Asp Phe Leu Lys Gln Ala Glu Asp Trp Leu Ser Ser Leu Ala130 135 140 Arg Pro Gly Glu Thr Asp Gly Gly Asp Gly Leu Leu Gly Tyr AlaVal 145 150 155 160 Ala Leu Gly Lys Leu Leu Gln Trp Asp Arg Ser Ala LeuLys Asp Ile 165 170 175 Lys Val Pro Thr Leu Val Ile Trp Gly Asp Asp AspPro Leu Val Pro 180 185 190 Leu Lys Ala Ser Glu Lys Leu Ser Ala Leu PhePro Asn Ala Glu Val 195 200 205 Val Val Ile Asp Asp Ala Gly His Leu AlaLeu Leu Glu Lys Pro Glu 210 215 220 Glu Val Ala Glu Leu Ile Lys Phe LeuAla Leu Ser Thr Asn Asx Ile 225 230 235 240 Arg Asp Ala Leu Ser Thr AsnAsx Ile Arg Asp 245 250

That which is claimed:
 1. An isolated nucleic acid molecule selectedfrom the group consisting of: a) a nucleic acid molecule comprising anucleotide sequence having at least 85% sequence identity to thenucleotide sequence of SEQ ID NO:2, or the nucleotide sequence of thecDNA insert of the plasmid deposited with ATCC as Accession NumberPTA-1643, wherein said nucleotide sequence encodes a polypeptide havingcarboxypeptidase activity; b) a nucleic acid molecule comprising afragment of at least 50 contiguous nucleotides of the nucleotidesequence of SEQ ID NO:2, or the nucleotide sequence of the cDNA insertof the plasmid deposited with ATCC as Accession Number PTA-1643, c) anucleic acid molecule which encodes a fragment of a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, or the amino acidsequence encoded by the cDNA insert of the plasmid deposited with theATCC as Accession Number PTA-1643, wherein the fragment comprises atleast 15 contiguous amino acids of SEQ ID NO:2, or the amino acidsequence encoded by the cDNA insert of the plasmid deposited with theATCC as Accession Number PTA-1643, d) a nucleic acid molecule whichencodes a polypeptide having carboxypeptidase activity comprising theamino acid sequence of SEQ ID NO:1, or the amino acid sequence encodedby the cDNA insert of the plasmid deposited with the ATCC as AccessionNumber PTA-1643, wherein the nucleic acid molecule hybridizes to anucleic acid molecule comprising the complement of SEQ ID NO:2 understringent conditions; and e) a nucleic acid molecule comprising thecomplement of a), b), c), or d).
 2. The isolated nucleic acid moleculeof claim 1, comprising a nucleotide sequence having at least 95%sequence identity to the nucleotide sequence of SEQ ID NO:2, or thenucleotide sequence of the cDNA insert of the plasmid deposited withATCC as Accession Number PTA-1643, wherein said nucleotide sequenceencodes a polypeptide having carboxypeptidase activity, or a complementthereof.
 3. The nucleic acid molecule of claim 1 further comprisingvector nucleic acid sequences.
 4. The nucleic acid molecule of claim 1further comprising nucleic acid sequences encoding a heterologouspolypeptide.
 5. A host cell which contains the nucleic acid molecule ofclaim
 3. 6. The host cell of claim 5 which is a mammalian host cell. 7.A non-human mammalian host cell containing the nucleic acid molecule ofclaim
 1. 8. An isolated polypeptide selected from the group consistingof: a) a polypeptide having carboxypeptidase activity which is encodedby a nucleic acid molecule comprising a nucleotide sequence having atleast 85% identity to a nucleic acid comprising the nucleotide sequenceof SEQ ID NO:2, or the nucleotide sequence of the cDNA insert of theplasmid deposited with ATCC as Accession Number PTA-1643; b) apolypeptide having carboxypeptidase activity, wherein the polypeptide isencoded by a nucleic acid molecule which hybridizes to a nucleic acidmolecule comprising the complement of SEQ ID NO:2, or the nucleotidesequence of the cDNA insert of the plasmid deposited with ATCC asAccession Number PTA-1643 under stringent conditions; c) a fragment of apolypeptide comprising the amino acid sequence of SEQ ID NO: 1, or theamino acid sequence encoded by the cDNA insert of the plasmid depositedwith the ATCC as Accession Number PTA-1643, wherein the fragmentcomprises at least 15 contiguous amino acids of SEQ ID NO: 1; and d) apolypeptide having at least 85% sequence identity to the amino acidsequence SEQ ID NO: 1, wherein the polypeptide has carboxypeptidaseactivity.
 9. The isolated polypeptide of claim 8 comprising the aminoacid sequence of SEQ ID NO:1.
 10. The polypeptide of claim 8 furthercomprising heterologous amino acid sequences.
 11. An antibody whichselectively binds to a polypeptide of claim
 8. 12. A method forproducing a polypeptide selected from the group consisting a) apolypeptide comprising a fragment of the amino acid sequence of SEQ IDNO: 1, or the amino acid sequence encoded by the cDNA insert of theplasmid deposited with the ATCC as Accession Number PTA-1643, whereinthe fragment comprises at least 15 contiguous amino acids of SEQ IDNO:1, or the amino acid sequence encoded by the cDNA insert of theplasmid deposited with the ATCC as Accession Number PTA-1643; c) apolypeptide having carboxypeptidase activity, wherein the polypeptide isencoded by a nucleic acid molecule which hybridizes to a nucleic acidmolecule comprising the complement of SEQ ID NO:2, or the nucleotidesequence of the cDNA insert of the plasmid deposited with ATCC asAccession Number PTA-1643, d) a polypeptide having at least 85% sequenceidentity to the amino acid sequence of SEQ ID NO:2, wherein saidpolypeptide has carboxypeptidase activity; comprising culturing the hostcell of claim 5 under conditions in which the nucleic acid molecule isexpressed.
 13. A method for detecting the presence of a polypeptide ofclaim 8 in a sample, comprising: a) contacting the sample with acompound which selectively binds to a polypeptide of claim 8; and b)determining whether the compound binds to the polypeptide in the sample.14. The method of claim 13, wherein the compound which binds to thepolypeptide is an antibody.
 15. A kit comprising a compound whichselectively binds to a polypeptide of claim 8 and instructions for use.16. A method for detecting the presence of a nucleic acid molecule ofclaim 1 in a sample, comprising the steps of: a) contacting the samplewith a nucleic acid probe or primer which selectively hybridizes to thenucleic acid molecule; and b) determining whether the nucleic acid probeor primer binds to a nucleic acid molecule in the sample.
 17. The methodof claim 16, wherein the sample comprises mRNA molecules and iscontacted with a nucleic acid probe.
 18. A kit comprising a compoundwhich selectively hybridizes to a nucleic acid molecule of claim 1 andinstructions for use.
 19. A method for identifying a compound whichbinds to a polypeptide of claim 8 comprising the steps of: a) contactinga polypeptide, or a cell expressing a polypeptide of claim 8 with a testcompound; and b) determining whether the polypeptide binds to the testcompound.
 20. The method of claim 19, wherein the binding of the testcompound to the polypeptide is detected by a method selected from thegroup consisting of: a) detection of binding by direct detecting of testcompound/polypeptide binding; b) detection of binding using acompetition binding assay; c) detection of binding using an assay forcarboxypeptidase activity.
 21. A method for modulating the activity of apolypeptide of claim 8 comprising contacting a polypeptide or a cellexpressing a polypeptide of claim 8 with a compound which binds to thepolypeptide in a sufficient concentration to modulate the activity ofthe polypeptide.
 22. A method for identifying a compound which modulatesthe activity of a polypeptide of claim 8, comprising: a) contacting apolypeptide of claim 8 with a test compound; and b) determining theeffect of the test compound on the activity of the polypeptide tothereby identify a compound that modulates the activity of thepolypeptide.